Methods for treating pain

ABSTRACT

The present invention features methods and compositions for preventing, reducing, or treating a traumatic, metabolic or toxic peripheral nerve lesion or pain including, for example, neuropathic pain, inflammatory and nociceptive pain by administering to a mammal in need thereof a compound that reduces the expression or activity of BH4. According to this invention, this reduction may be achieved by reducing the enzyme activity of any of the BH4 synthetic enzymes, such as GTP cyclohydrolase (GTPCH), sepiapterin reductase (SPR), or dihydropteridine reductase (DHPR); by antagonizing the cofactor function of BH4 on BH4-dependent enzymes; or by blocking BH4 binding to membrane bound receptors. The compounds of the invention may be administered alone or in combination with a second therapeutic agent. 
     The invention also provides methods for diagnosing pain or a peripheral nerve lesion in a mammal by measuring the levels of BH4 or its metabolites in biological sample. Alternatively, pain or a peripheral nerve lesion may be diagnosed by measuring the levels or activity of any one of the BH4 synthetic enzymes in tissue samples of a mammal. Also disclosed are screening methods that make use of BH4 or BH4 synthetic enzymes, BH4-dependent enzymes, and BH4-binding receptors for the identification of novel therapeutics for the treatment, prevention, or reduction of pain.

This application claims benefit of the filing date of the copending U.S.Provisional Application No. 60/520,536, filed Nov. 13, 2003, herebyincorporated by reference.

FIELD OF THE INVENTION

In general, the invention features methods for diagnosing, treating,reducing, or preventing pain.

BACKGROUND OF THE INVENTION

The sensation of pain is a common symptom that may be indicative of anunderlying disease or injury, or the expression of an abnormal functionwithin the nervous system. Pain is often the primary incentive for whichtreatment is sought.

Pain can take a variety of forms depending on its origin. Pain may bedescribed as being peripheral neuropathic if the initiating injuryoccurs as a result of a complete or partial transection of a nerve ortrauma to a nerve plexus. Alternatively, pain is described as beingcentral neuropathic following a lesion to the central nervous system,such as a spinal cord injury or a cerebrovascular accident. Inflammatorypain is a form of pain that is caused by tissue injury or inflammation(e.g., in postoperative pain or rheumatoid arthritis). Following aperipheral nerve injury, symptoms are typically experienced in a chronicfashion, distal to the site of injury and are characterized byhyperesthesia (enhanced sensitivity to a natural stimulus), hyperalgesia(abnormal sensitivity to a noxious stimulus), allodynia (widespreadtenderness, associated with hypersensitivity to normally innocuoustactile stimuli), and/or spontaneous burning or shooting lancinatingpain. In inflammatory pain, symptoms are apparent, at least initially,at the site of injury or inflamed tissues and typically accompanyarthritis-associated pain, musculo-skeletal pain, and postoperativepain. Nociceptive pain is the pain experienced in response to a noxiousstimulus, such as a needle prick or during trauma or surgery. Functionalpain refers to conditions in which there is no obvious peripheralpathology or lesion to the nervous system. This particular form of painis generated by abnormal function of the nervous system and conditionscharacterized by such pain include fibromyalgia, tension-type headache,and irritable bowel syndrome. The different types of pain may coexist orpain may be transformed from inflammatory to neuropathic during thenatural course of the disease, as in post-herpetic neuralgia.

Although one approach for the treatment of pain is the removal of thecausative or etiological agent (disease modifying therapy), the painoften outlasts the duration of the initiating cause. Accordingly,symptomatic control is essential. In cases in which the sensation ofpain becomes unbearable, rapid and effective analgesia is imperative(e.g., postoperative state, burns, trauma, cancer, and sickle cellcrisis). Currently, there exist a wide variety of analgesic agentsuseful for the management of pain, including for example non-steroidalanalgesic agents (NSAIDs), anticonvulsants, and opioid analgesics.Despite their efficacy, the chronic use of such agents is often notrecommended because of the potential debilitating side effects, such asgastric irritation, toxicity to the liver, respiratory depression,sedation, psychotomimetic effects, constipation, nausea, tolerance,dependence, and the risk of abuse. Also, these agents are sometimessuboptimal, particularly for neuropathic and functional pain.

Thus, better therapeutic strategies are required for the treatment andmanagement of pain.

SUMMARY OF THE INVENTION

In general, the present invention features methods for the diagnosis,treatment, reduction, and prevention of pain or of endogenous mechanismsthat further increase a traumatic, metabolic or toxic peripheral nervelesion in a mammal. According to this invention, a mammal (e.g., ahuman) is administered a composition (e.g., methotrexate) that reducestetrahydrobiopterin (BH4) biological activity such that pain is reduced,prevented, or treated. Alternatively, pain may be reduced in the mammalbeing treated by decreasing the levels or activity of any one of theenzymes involved in the synthesis of BH4, i.e. BH4 synthetic enzymes. Inthis regard, BH4 synthesis may be reduced by decreasing the biologicalactivity of at least one, two, three, or more than three of thefollowing enzymes: sepiapterin reductase (SPR), PyruvoyltetrahydropterinSynthase (PTPS), GTP cyclohydrolase (GTPCH), Pterin-4α-carbinolaminedehydratase, and dihydropteridine reductase (DHPR). Such activity may bereduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%,99%, or even 100% relative to an untreated control. Alternatively, BH4biological activity may be reduced by increasing the expression orGTPCH-binding or other biological activity of GTP cyclohydrolasefeedback regulatory protein (GFRP). GFRP biological activity may beincreased by administering a BH4 or phenylalanine analog thatspecifically binds to GFRP or a GTPCH:GFRP complex. Traumatic nervelesions include those caused by mechanical insults and compressiveinjuries. Compressive injuries may be caused by external trauma orinjury, or from internal conditions and disease states, such as acompression resulting from an infiltrative tumor. Metabolic nervelesions amenable to treatment using the methods of this inventioninclude, for example, diabetic peripheral neuropathies, heritableneuropathies, or neuropathies caused by infectious agents such as thehuman immunodeficiency virus (HIV). Toxic nerve lesions include, forexample, those cause by other therapeutic agents (e.g.,chemotherapeutics), or chemicals and environmental toxicants (e.g.,heavy metals and organic solvents).

According to this invention, a reduction in pain may result, forexample, from changes in the function of primary sensory neurons orneurons within the dorsal horn of the spinal cord, in the brainstem, orin the brain. Such changes may result, for example, from a reduction inthe synthesis of BH4, leading to a reduction in the activity of variousenzymes (e.g., nitric oxide synthase) that utilize BH4 as a cofactor andto a reduction in the activation of membrane-bound BH4-binding receptorsfollowing its release from cells. BH4 action on BH4-binding receptorsmay be inhibited or reduced by means of competitive or non-competitiveBH4-like receptor antagonists. BH4 action on enzymes which use BH4 as acofactor may be inhibited by means of BH4-like competitive antagonists.Such binding or activity may be reduced by at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% 95%, 99%, or even 100% relative to an untreatedcontrol.

Furthermore, levels of tetrahydrobiopterin (BH4), its precursors, or itsmetabolites may be measured in a biological sample obtained from amammal (e.g., plasma, tissue sample, cerebrospinal fluid, synovialfluid, or tissue exudates) and, in turn, serve as diagnostic tools andas biomarkers of pain or nerve injury. Methods for measuring BH4 aredescribed, for example, in Powers et al. (1988) J Chromatogr432:321-328; Blau et al. (1994) Clin Chim Acta 226:159-169; Ponzone etal. (1994) Eur J Pediatr 153:616; Zorzi et al. (2002) Mol Genet Metab75:174-177; and Shiraki et al. (1994) Eur J Pediatr 153:616. Optionally,an increase in levels or activities of any one of the BH4 syntheticenzymes may also diagnose pain in a mammal.

The methods of this invention are therefore useful for the diagnosis,treatment, reduction, or prevention of various forms of pain, forexample, nociceptive pain, inflammatory pain, functional pain andneuropathic pain, all of which may be acute or chronic. Thus, the mammalbeing treated may be diagnosed as having peripheral diabetic neuropathy,compression neuropathy, post herpetic neuralgia, trigeminal orglossopharyngeal neuralgia, post traumatic or post surgical nervedamage, lumbar or cervical radiculopathy, AIDS neuropathy, metabolicneuropathy, drug induced neuropathy, complex regional pain syndrome,arachnoiditis, spinal cord injury, bone or joint injury, tissue injury,psoriasis, scleroderma, pruritis, cancer (e.g., prostate, colon, breast,skin, hepatic, or kidney), cardiovascular disease (e.g., myocardialinfarction, angina, ischemic or thrombotic cardiovascular disease,peripheral vascular occlusive disease, or peripheral arterial occlusivedisease), sickle cell anemia, migraine cluster or tension-typeheadaches, inflammatory conditions of the skin, muscle, or joints,fibromyalgia, irritable bowel syndrome, non cardiac chest pain,cystitis, pancreatitis, or pelvic pain. Alternatively, the pain forwhich treatment is being sought may be the result of a traumatic injury,surgery, burn of the cutaneous tissue (caused by a thermal, chemical, orradiation stimulus), or a sunburn.

According to this invention, the mammal being treated may beadministered with a composition containing, for example, at least one ofthe following compounds: methotrexate, trimethoprim-sulfamethoxazole,2,6 diamino hydroxypyrimidine (DAHP), Tetrahydro-L-biopterin,L-Sepiapterin, 7,8-dihydro-L-Biopterin, 6,7-dimethyltetrahydropterinhydrochloride, 8-bromo-cGMP, N-acetyl-serotonin (NAS),N-Chloroacetylserotonin, N-Methoxyacetylserotonin, andN-Chloroacetyldopamine.

Alternatively, the composition of the invention may contain a compoundhaving the formula:

such that R¹ is a H, C₁₋₆ alkyl, halo, NO₂, CN, CO₂R⁴, CONR⁴R⁵, SO₂R⁴,SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵. Each of R⁴ and R⁵ may be, independently, a H,C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl;R² may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl; and R³ may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶, CONR⁷R⁸, SO₂R⁶, or SO₂NR⁷R⁸,where R⁶ is a C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl, and each of R⁷ and R⁸ is, independently, a H, C₁₋₆ alkyl,C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl.

Alternatively, R³ may be as above and R¹ and R² together may berepresented by

where the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Optionally, R³ may be as above and R¹ and R² together may be representedby

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² or R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, such that each of R⁷ and R⁸ is,independently, a H, C₁-C₆ alkyl, C₆-C₁₂ aryl, heteroaryl, C₁-C₄ alkaryl,or C₁-C₄ alkheteroaryl.

As another alternative, R¹ and R² together may be represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing the R¹⁴ and thenitrogen bearing R².

Optionally, the composition may have be a compound of formula (I), suchthat R¹ is a H, C₁₋₆ alkyl, halo, NO₂, CN, CO₂R⁴, CONR⁴R⁵, SO₂R⁴,SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵. Accordingly, each of R⁴ and R⁵ may be,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl, R² may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, and R³ may be a H, C₁₋₆ alkyl,C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶,CONR⁷R⁸, SO₂R⁶, or SO₂NR⁷R⁸, where R⁶ is a C₁₋₆ alkyl, C₆₋₁₂ aryl,heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl and each of R⁷ and R⁸may be, independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl.

If desired, the composition contains a compound of formula:

in which R¹ and R² together may be represented by

such that the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R³ is as above and R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² and R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, where each of R⁴, R⁷ and R⁸ is,independently, a H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R¹ and R² together may be represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².

If the composition contains a compound of formula:

R¹ and R² together may be represented by

where the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R³ is as above and R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² and R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, such that each of R⁷ and R⁸ is,independently, H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl.

Optionally, the composition may contain a compound of formula:

in which R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².

Exemplary compounds that may be contained within the composition of theinvention include, for example:

Furthermore, the composition of the invention may contain a compoundhaving the formula:

in which R¹⁵ is a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl,or C₁₋₄ alkheteroaryl and R¹⁶ is a H, C₁₋₆ alkyl, C₆₋₁₂ aryl,heteroaryl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R¹⁷, CONR¹⁸R¹⁹, SO₂R¹⁷,or SO₂NR¹⁸R¹⁹. R¹⁷ may be a C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl and each of R¹⁸ and R¹⁹ may be,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

If desired, a second therapeutic agent, such as an analgesic agent, maybe administered in combination with the composition of the invention,including for example, a non-steroidal anti-inflammatory agent (NSAIDs)(including acetaminophen, non-COX-2 selective agents, or COX-2 selectiveinhibitory drugs), opioid receptor agonist (e.g., morphine, codeine,hydrocodone, hydromorphone, levorphanol, methadone, meperidine,butorphanol, bupranorphine, nalbuphine, alfentanil, sufentanil,fentanyl, tramadol, pentazocine, propoxyphene, or oxycodone), tricyclicantidepressant (e.g., doxepin, amitriptyline, imipramine, nortriptyline,desipramine, or venlafaxine), SSRIs (e.g. paroxetine, sertraline,fluoxetine, or citalopram), anticonvulsants (e.g., phenytoin,carbamazepine, oxcarbazepine, lamotrigine, valproate, pregabalin, orgabapentin), voltage-gated sodium channel blockers, membranestabilizers, nerve blockers (e.g. lidocaine, bupivicaine, prilocaine, ormexilitine), N-type calcium channel blockers (e.g ziconitide), serotoninreceptor agonists such as 5-HT1D receptor agonists (e.g. sumatriptam,zolmatriptan, rizotriptan, naratriptan, almotriptan, or frovatriptan),steroids (e.g. cortisone, hydrocrtisone, prednisolone, ormethylprednisolone). According to this invention, the second therapeuticagent may or may not have a therapeutic effect (such as analgesia) whenadministered as a single agent but results in such an effect (or anadditive or synergistic increase) when administered in combination withthe composition of the invention. Other exemplary analgesic agentsuseful in the invention may include, for example, acetaminophen,acetylsalicylic acid, ibuprofen, naproxen, fenoprofen, indomethacin,ketorolac, rofecoxib, celecoxib, valdecoxib, paracoxib, clonazepam,diazepam, capsaicin, ketamine, clonidine, or baclofen.

Alternatively, the second therapeutic agent may be an inhibitor of anyenzyme which utilizes BH4 as a cofactor including any of the followingenzymes: all isoforms of nitric oxide synthase (NOS) such as eNOS, iNOS,or nNOS; tyrosine hydroxylase; tryptophan hydroxylase I (non-neuronalTph1) and II (neuronal Tph2); phenylalanine hydroxylase;dopamine-β-hydroxylase; N-methyltransferase; and ether lipid oxidase.The second therapeutic agent may also include agents that inhibit directeffects of BH4 independent of its co-enzyme function, such as agentsthat interfere with BH4 binding to membrane bound receptors.

The composition of the invention and the second therapeutic agent may beadministered together (as two separate formulations or a singleformulation) or separately (e.g., within one hour, two hours, threehours, six hours, or twenty four hours of each other).

In another aspect, the invention features a method for diagnosing painor a traumatic, metabolic or toxic peripheral nerve lesion in a mammalby measuring the levels of BH4, BH4 precursors, or intermediates (e.g.,7,8-dihydroneopterin triphosphate and 6-pyruvoyl tetrahydropterin), orBH4 metabolites (e.g., pterin, bipterin, 7,8 dihydropterin, 7,8dihydroxanthopterin, xanthopterin, isoxanthopterin, leucopterin, orneopterin) in a biological sample of a mammal (e.g., in the serum,plasma, urine, cerebrospinal fluid, synovial fluid, tissue exudates, ortissue samples). According to this aspect of the invention, levels ofBH4, BH4 precursors, BH4 intermediates, or BH4 metabolites serve asbiomarkers of pain such that an increase in any of these moleculesdiagnoses pain in the mammal. Alternatively, the clinical diagnosis ofpain may be supported in a mammal by measuring the level (e.g., mRNA orprotein levels) or activity of any one of the BH4 synthetic enzymes(e.g., sepiapeterin reductase (SPR), Pyruvoyltetrahydropterin Synthase(PTPS), GTP cyclohydrolase (GTPCH), Pterin-4α-carbinolamine dehydratase,and dihydropteridine reductase (DHPR)) in the mammal. Similarly, thepain diagnosis is supported by determining an increase in the activityor level of such enzymes.

The present invention also provides a method for identifying a candidatecompound for treating, reducing, or preventing pain or endogenousmechanisms that further increase a traumatic, metabolic or toxicperipheral nerve lesion in a mammal. The method involves the steps of:(a) contacting a cell synthesizing BH4 with a candidate compound; and(b) measuring the BH4 level or activity (e.g., ability to function as aco-factor or ability to bind membrane-bound receptors). A compound thatdecreases the level or activity of BH4 relative to the BH4 level oractivity in a cell not contacted with the compound is identified as acandidate compound for treating, reducing, or preventing pain or atraumatic, metabolic or toxic peripheral nerve lesion in a mammal.Alternatively, the cell in step (a) may express any one of the BH4synthetic enzymes (e.g., sepiapeterin reductase (SPR),Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase (GTPCH),Pterin-4α-carbinolamine dehydratase, and dihydropteridine reductase(DHPR)) and, optionally, step (b) involves measuring the expression orbiological activity of such enzymes to assess the inhibitory activity ofthe candidate compound. If desired, the BH4 synthetic enzyme may be aprotein fusion gene. In preferred embodiments, step (b) involves themeasurement of BH4 levels or activity, or alternatively, the measurementof the mRNA or protein levels or enzyme activity of one of the BH4synthetic enzymes. Preferably, the cell is a mammalian cell (e.g., ahuman or rodent cell).

In a related aspect, the invention provides an alternative method foridentifying a candidate compound for treating, reducing, or preventingpain or a traumatic, metabolic or toxic peripheral nerve lesion in amammal. This method involves the steps of: (a) identifying a pteridineor specific BH4 binding site on a protein (e.g., a BH4-binding receptoror BH4-dependent enzyme); (b) contacting such a BH4 binding protein(receptor or enzyme) with a candidate compound; and (c) determiningwhether the candidate compound binds to the BH4 site on the protein andinhibits BH4 binding or activity on the protein (for example, by itselfbinding to the BH4 site). Compounds that inhibit BH4 activity or bindingare identified as candidate compounds for treating, reducing, orpreventing pain or a a traumatic, metabolic or toxic peripheral nervelesion. Optionally, this method may further involve contacting thecandidate compound with any one of the enzymes that use BH4 as acofactor (e.g., NOS) or membrane receptors that bind BH4 and determiningwhether the candidate compound binds and/or inhibits the activity ofsuch an enzyme or receptor.

The invention also provides yet another method for identifying acandidate compound for treating, reducing, or preventing pain or atraumatic, metabolic or toxic a peripheral nerve lesion in a mammal.This method involves the steps of: (a) providing GTP cyclohydrolase(GTPCH), GTPCH Feedback Regulatory Protein (GFRP), and a candidatecompound; (b) measuring the binding of the GTPCH and the GFRP; and (c)identifying a candidate compound as useful for treating, reducing, orpreventing pain or a traumatic, metabolic or toxic peripheral nervelesion, wherein the binding of the GTPCH and GFRP is increased in thepresence of the candidate compound. In preferred embodiments, the GTPCHand GFRP are human proteins. In this method, the candidate compound maypreferably bind to either GFRP or the GTPCH:GFRP complex.

In preferred embodiments, the method also tests the ability of thecandidate compound to reduce the expression of one or all of the BH4synthetic enzymes in a cell, for example, a mammalian cell such as arodent or human cell leading to a reduction in BH4 levels. Mostpreferably, the BH4 synthetic enzyme gene is human sepiapterin reductase(SPR), Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase I(GTPCH I), Pterin-4α-carbinolamine dehydratase, or dihydropteridinereductase (DHPR).

The present invention further includes kits for carrying out the methodsof the invention. For example, the invention includes a kit containing acomposition that reduces the level and activity of tetrahydrobiopterin(BH4) in an amount sufficient to treat, reduce, or prevent pain or atraumatic, metabolic or toxic peripheral nerve lesion as well asinstructions for delivery of the composition to a mammal for treating,reducing, or preventing pain or a traumatic, metabolic or toxicperipheral nerve lesion.

The present invention further includes a diagnostic kit for themeasurement of BH4, its precursors and intermediates (e.g.,7,8-dihydroneopterin triphosphate and 6-pyruvoyl tetrahydropterin), ormetabolites (e.g., pterin, biopterin, 7,8 dihydropterin, 7,8dihydroxanthopterin, xanthopterin, isoxanthopterin, leucopterin, andneopterin) from a biological sample of a mammal (e.g., serum, plasma,urine, cerebrospinal fluid, synovial fluid, tissue exudates, and tissuesamples). For example, the invention includes a kit containing anantibody that is specific to BH4 as well as instructions for diagnosingpain in a mammal.

The invention also features methods for identifying a BH4 targetprotein. The first method consists of the steps of (a) providing asample and BH4; (b) contacting the sample with the BH4 under conditionsthat allow binding between the sample proteins and the BH4; and (c)assessing the binding of the BH4 to a sample protein by detecting theBH4, wherein a sample protein that binds to BH4 is identified as a BH4target protein. In one embodiment, the assessing step (c) uses adetection method such as mass spectrometry, surface plasmon resonancemicroscopy, or atomic force microscopy. Biological samples which can beused in this method to identify BH4 target proteins may come from anysource. Preferred samples are extracts prepared from mammalian nervoustissue such as, for example, nervous tissue containing the dorsal hornor the dorsal root ganglia. Preferably, samples contain membrane-boundproteins.

A second method for identifying a BH4 target protein contains the stepsof (a) providing BH4 and an array, wherein the array consists of aplurality of immobilized purified protein species, wherein each of theprotein species in the array is spatially separated from each of theother protein species; (b) contacting the array with the BH4 underconditions that allow binding between the protein species and the BH4;and (c) assessing the binding of the BH4 to the protein species, whereina protein species that binds to the BH4 is identified as a BH4 targetprotein.

In preferred embodiments of either of the two foregoing methods, the BH4is detectably labeled. Useful detectable labels include, for example, aradioisotope (e.g., tritium) or biotin. In other useful embodiments, theassessing step (c) requires the use of a BH4-specific antibody.

Finally, the invention features compositions for reducing BH4 biologicalactivity.

Compositions of the invention may have the formula:

such that R¹ is a H, C₁₋₆ alkyl, halo, NO₂, CN, CO₂R⁴, CONR⁴R⁵, SO₂R⁴,SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵. Each of R⁴ and R⁵ may be, independently, a H,C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl;R² may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl; and R³ may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶, CONR⁷R⁸, SO₂R⁶, or SO₂NR⁷R⁸,where R⁶ is a C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl, and each of R⁷ and R⁸ is, independently, a H, C₁₋₆ alkyl,C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl.

Alternatively, R³ may be as above and R¹ and R² together may berepresented by

where the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Optionally, R³ may be as above and R¹ and R² together may be representedby

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² or R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, such that each of R⁷ and R⁸ is,independently, a H, C₁-C₆ alkyl, C₆-C₁₂ aryl, heteroaryl, C₁-C₄ alkaryl,or C₁-C₄ alkheteroaryl.

As another alternative, R¹ and R² together may be represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing the R¹⁴ and thenitrogen bearing R².

Optionally, the composition may have be a compound of formula (I), suchthat R¹ is a H, C₁₋₆ alkyl, halo, NO₂, CN, CO₂R ⁴, CONR⁴R⁵, SO₂R⁴,SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵. Accordingly, each of R⁴ and R⁵ may be,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl, R² may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, and R³ may be a H, C₁₋₆ alkyl,C₆₋₁₂ aryl, heteroaryl, alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶, CONR⁷R⁸,SO₂R⁶, or SO₂NR⁷R⁸, where R⁶ is a C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl and each of R⁷ and R⁸ may be,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

If desired, the composition contains a compound of formula:

in which R¹ and R² together may be represented by

such that the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R³ is as above and R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² and R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, where each of R⁴, R⁷ and R⁸ is,independently, a H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R¹ and R² together may be represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².

If the composition contains a compound of formula:

R¹ and R² together may be represented by

where the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R³ is as above and R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² and R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, such that each of R⁷ and R⁸ is,independently, H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl.

Optionally, the composition may contain a compound of formula:

in which R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².

Exemplary compounds that may be contained within the composition of theinvention include, for example:

Furthermore, the composition of the invention may contain a compoundhaving the formula:

in which R¹⁵ is a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl,or C₁₋₄ alkheteroaryl and R¹⁶ is a H, C₁₋₆ alkyl, C₆₋₁₂ aryl,heteroaryl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R¹⁷, CONR¹⁸R¹⁹, SO₂R¹⁷,or SO₂NR¹⁸R¹⁹. R¹⁷ may be a C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl and each of R¹⁸ and R¹⁹ may be,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

The compositions of the invention may be combined with any secondtherapeutic suitable for use in the above-described methods. Thecompositions alone or in combination with any second therapeutic may bepresent in a therapeutic composition in association with apharmaceutically acceptable carrier or excipient.

By “BH4 synthetic enzyme fusion gene” is meant a promoter and/or all orpart of a coding region of a BH4 synthetic enzyme (e.g., sepiapeterinreductase (SPR), Pyruvoyltetrahydropterin Synthase (PTPS), GTPcyclohydrolase (GTPCH), Pterin-4α-carbinolamine dehydratase, anddihydropteridine reductase (DHPR)) operably linked to a second,heterologous nucleic acid sequence. In preferred embodiments, thesecond, heterologous nucleic acid sequence is a reporter gene, that is,a gene whose expression may be assayed; exemplary reporter genesinclude, without limitation, those genes encoding glucuronidase (GUS),luciferase, chloramphenicol transacetylase (CAT), green fluorescentprotein (GFP), alkaline phosphatase, and (3-galactosidase.

By a “candidate compound” is meant a chemical, be it naturally-occurringor artificially-derived. Candidate compounds include, for example,peptides, polypeptides, synthetic organic molecules, naturally occurringorganic molecules, nucleic acids, peptide nucleic acids, and componentsthereof.

By “dominant negative protein” is meant any polypeptide having at least50%, 70%, 80%, 90%, 95%, or even 99% sequence identity to 10, 20, 35,50, 100, 150, or more than 150 amino acids of the wild type protein towhich the dominant negative protein corresponds. In addition toinactivating mutations, dominant negative proteins may consist ofdeletions or truncations of a wild-type molecule. For example, adominant negative BH4 synthetic enzyme may be a truncated BH4 syntheticenzyme mutant that has a deletion such that it no longer functions toproduce BH4 or its intermediates (e.g., 7,8-dihydroneopterintriphosphate and 6-pyruvoyl tetrahydropterin) including, for example, aGTPCH that has lost its catalytic activity, thereby disrupting the BH4synthetic pathway.

By “opioid receptor agonist” is meant any naturally occurring,semi-synthetic, or synthetic compound that binds to the mu, kappa, ordelta opioid receptor subtypes and mimics the function of opioids atthese receptors. The opioid receptor agonist may be a peptide or anon-peptide compound. Preferably, opioid receptor agonists have a K_(d)for at least one opioid receptor subtype of <1 μM, more preferably <100nM, most preferably <10 nM, or even <1 nM. Opioid receptor agonistsinclude generally, for example, members from the phenanthrene, phenylheptylamine, phenylpiperidine, morphinan, and benzomorphan chemicalfamilies. Opioid receptor agonists include, for example, morphine,hydormorphone, oxymorphone, codeine, oxycodone, hydrocodone, methadone,meperidine, levorphanol, nalbuphine, sufentanil, alfentanil,buprenorphine, pentazocine, propoxyphene and butorphanol.

By “an effective amount” is meant an amount of a compound, alone or aspart of a combination according to the invention, required to prevent,reduce, or eliminate the sensation of pain. The effective amount ofactive compound(s) used to practice the present invention fortherapeutic treatment of pain varies depending upon the manner ofadministration, the age, and body weight, of the subject as well as theunderlying pathology that is causing the pain. Ultimately, the attendingphysician or veterinarian will decide the appropriate amount and dosageregimen. Such amount is referred to as an “effective” amount.

By “pain” is meant all types of pain including, for example, peripheraland central neuropathic pain, functional pain, inflammatory pain ornociceptive pain, whether acute or chronic. Exemplary pain conditionsinclude post-operative or post-traumatic pain, chronic lower back pain,pain of rheumatoid arthritis, osteoarthritis, fibromyalgia, clusterheadaches, post-herpetic neuralgia, phantom limb pain, central strokepain, dental pain, opioid-resistant pain, visceral pain, bone injurypain, labor pain, pain resulting from burns including sunburns,post-partum pain, migraine, tension type headache, angina pain, andgenitourinary tract-related pain (e.g., cystitis).

By “reduce the tetrahydrobiopterin (BH4) biological activity” is meantto reduce a functional outcome associated with a biological activityattributed to BH4. Generally, the reduction of BH4 biological activitymay be the result of, for example, a reduction in the amount (level) ofthe BH4 molecules and may be affected by reducing/inhibiting de novo BH4synthesis, increasing/accelerating BH4 catabolism, or a combination ofthe two.

Alternatively, the biological activity of BH4 may be reduced byinhibiting the effect of BH4 at a target molecule. For example, acompetitive BH4 inhibitor that binds to a BH4 binding site on aneffector protein (e.g, an enzyme) will reduce the biological activity.Non-competitive BH4 inhibitors are also included in this definition.Likewise, a BH4 binding molecule which effectively sequesters BH4 andprevents it from binding to an effector molecule also has the effect ofreducing BH4 biological activity. Such reduction may be, for example, adecrease of least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, orgreater than 100%, relative to control conditions. More specifically,BH4 level or activity may be decreased, for example, by reducing theenzyme activity of enzymes involved in the BH4 synthesis pathway, suchas GTP cyclohydrolase (GTPCH), Sepiapterin Reductase (SPR), andDihydropteridine reductase (DHPR). Preferably, such enzyme activity isreduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,or even greater than 100%, relative to a control. GTPCH may, forexample, be inhibited by increasing the levels or binding activity ofGTPCH Feedback Regulatory Protein (GFRP).

By “treating, reducing, or preventing pain” is meant preventing,reducing, or eliminating the sensation of pain in a subject before,during, or after it has occurred. As compared with an equivalentuntreated control, such reduction or degree of prevention is at least5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by anystandard technique known in the art. To treat pain, according to themethods of this invention, the treatment does not necessarily providetherapy for the underlying pathology that is causing the painfulsensation. Treatment of pain can be purely symptomatic.

By “diagnosing pain” is meant detecting pain caused by any stimulus in amammal including damage to peripheral nerves. For example, pain may bediagnosed by detecting a surrogate marker that is associated orcorrelated with the sensation of pain. According to the invention, painor a traumatic, metabolic or toxic peripheral nerve lesion is diagnosedby measuring and detecting an increase in the levels of BH4, BH4intermediates, BH4 precursors, or BH4 metabolites in a biological samplefrom the mammal (e.g., serum, plasma, urine, cerebrospinal fluid,synovial fluid, tissue exudates, or tissue samples). Pain is diagnosedin a mammal if such levels are increased by at least 10%, 20%, 30%, 40%,50%, 60%, 80%, 90%, 95%, 100, or more than 100% above a control.Alternatively, the levels or activity of any one of BH4 syntheticenzymes are measured and pain is diagnosed if an increase in the levelor activity of a BH4 enzyme is detected. Desirably, such increase is atleast 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, 100%, or more than100% above control conditions.

By “specific for” as used herein in reference to an antibody is meant anincreased affinity of an antibody for a particular protein or antigen,relative to an equal amount of any other protein or antigen. Forexample, an antibody (e.g., a human monoclonal antibody) that isspecific for BH4 desirably has an affinity for BH4 that is least 2-fold,5-fold, 10-fold, 30-fold, or 100-fold greater than for an equal amountof any other antigen, including related antigens. Binding of an antibodyto another protein or antigen may be determined as described herein, andby any number of standard methods in the art, e.g., Western analysis,ELISA, or co-immunoprecipitation.

By “substantially identical,” when referring to a protein orpolypeptide, is meant a protein or polypeptide exhibiting at least 75%,but preferably 85%, more preferably 90%, most preferably 95%, or even99% identity to a reference amino acid sequence. For proteins orpolypeptides, the length of comparison sequences will generally be atleast 20 amino acids, preferably at least 30 amino acids, morepreferably at least 40 amino acids, and most preferably 50 amino acidsor the full length protein or polypeptide. Nucleic acids that encodesuch “substantially identical” proteins or polypeptides constitute anexample of “substantially identical” nucleic acids; it is recognizedthat the nucleic acids include any sequence, due to the degeneracy ofthe genetic code, that encodes those proteins or polypeptides. Inaddition, a “substantially identical” nucleic acid sequence alsoincludes a polynucleotide that hybridizes to a reference nucleic acidmolecule under high stringency conditions.

By “high stringency conditions” is meant any set of conditions that arecharacterized by high temperature and low ionic strength and allowhybridization comparable with those resulting from the use of a DNAprobe of at least 40 nucleotides in length, in a buffer containing 0.5 MNaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (Fraction V), at atemperature of 65 C, or a buffer containing 48% formamide, 4.8×SSC, 0.2M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10% dextran sulfate, and 0.1%SDS, at a temperature of 42 C. Other conditions for high stringencyhybridization, such as for PCR, Northern, Southern, or in situhybridization, DNA sequencing, etc., are well known by those skilled inthe art of molecular biology. See, e.g., F. Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998,hereby incorporated by reference.

As used herein, the terms “alkyl” and the prefix “alk-” are inclusive ofboth straight chain and branched chain saturated or unsaturated groups,and of cyclic groups, including cycloalkyl and cycloalkenyl groups.Unless otherwise specified, acyclic alkyl groups contain 1 to 6 carbons.Cyclic groups can be monocyclic or polycyclic and preferably have 3 to 8ring carbon atoms. Exemplary cyclic groups include cyclopropyl,cyclopentyl, cyclohexyl, and adamantyl groups. The alkyl group may besubstituted or unsubstituted. Exemplary substituents include alkoxy,aryloxy, sulfhydryl, alkylthio, arylthio, halogen, hydroxyl,fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino,quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.

By “aryl” is meant a carbocyclic aromatic ring or ring system. Unlessotherwise specified, aryl groups contain 6 to 18 carbons. Examples ofaryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenylgroups.

By “heteroaryl” is meant an aromatic ring or ring system that containsat least one ring hetero-atom (e.g., O, S, Se. N, and P). Unlessotherwise specified, heteroaryl groups contain 1 to 9 carbons.Heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl,pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl,tetrazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl, pyrimidyl,pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole,indazolyl, indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl,cinnolinyl, quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl,purinyl, and carbazolyl groups.

By “heterocycle” is meant a non-aromatic ring or ring system thatcontains at least one ring heteroatom (e.g., O, S, Se, N, and P). Unlessotherwise specified, heterocyclic groups contain 2 to 9 carbons.Heterocyclic groups include, for example, dihydropyrrolyl,tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl,tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophene,tetrahydrothiophene, and morpholinyl groups.

Aryl, heteroaryl, or heterocyclic groups may be unsubstituted orsubstituted by one or more substituents including for example a C₁₋₆alkyl, hydroxy, halo, nitro, C₁₋₆ alkoxy, C₁₋₆alkylthio,trifluoromethyl, C₁₋₆ acyl, arylcarbonyl, heteroarylcarbonyl, nitrile,C₁₋₆ alkoxycarbonyl, alkaryl (in which the alkyl group has 1 to 6 carbonatoms), and alkheteroaryl (in which the alkyl group has 1 to 6 carbonatoms).

By “alkoxy” is meant a chemical substituent of the formula —OR, where Ris an alkyl group. By “aryloxy” is meant a chemical substituent of theformula —OR , where R is an aryl group. By “alkaryl” is meant a chemicalsubstituent of the formula —RR , where R is an alkyl group and R is anaryl group. By “alkheteraryl” is meant a chemical substituent of theformula -RR , where R is an alkyl group and R is a heteroaryl group.

By “halide” or “halogen” or “halo” is meant bromine, chlorine, iodine,or fluorine.

Overall, the present invention provides significant advantages overstandard therapies for the diagnosis, treatment, and prevention of pain.Based on our results, the administration of a therapeutic agent (e.g.,methotrexate) that reduces the level or activity of BH4 attenuates painin part by interfering with the activity of enzymes that utilize BH4 asa co-factor or membrane-bound receptors that bind to BH4 and modulateneuronal excitability or transmitter release. The present inventionfurther allows the diagnosis of pain in a mammal by measuring anddetecting an increase in the levels of BH4, BH4 intermediates, BH4metabolites, or BH4 precursors, or alternatively, by measuring anddetecting an increase in the activity or levels of any one the BH4synthetic enzymes. In addition, the candidate compound screening methodsprovided by the present invention allow for the identification of noveltherapeutics that modify the injury process and mitigate the symptoms byreducing the synthesis or action of BH4.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph representing the microarray expression profileanalysis of twenty-three genes, including Dihydropteridine reductase(DHPR), in the dorsal root ganglia (DRG) during development andadulthood, before and following peripheral nerve injury.

FIG. 1B represents a Northern slot blot analysis confirming mRNAregulation of DHPR in the dorsal root ganglia during development and inthe adult, before and following peripheral nerve injury.

FIG. 2A is a schematic diagram outlining the Tetrahydrobiopterin (BH4)synthesis pathway.

FIG. 2B is a schematic diagram showing the catabolism oftetrahydrobiopterin.

FIG. 3 is a table showing the change in expression levels of members ofthe BH4 synthetic pathway before and following peripheral nerve injurydetected by microarrays.

FIG. 4A is a picture of a Northern blot analysis showing mRNA expressionof GTP cyclohydrolase (GTPCH) in the dorsal root ganglia (DRG) before(naive) and following peripheral nerve injury.

FIG. 4B is a series of photomicrographs showing non-isotopic in situlocalization of GTPCH mRNA in the DRG before (naïve) and followingperipheral nerve injury.

FIG. 4C is a series of photomicrograph of DRGs showing increased GTPCHmRNA in neurons 3 days and 3 weeks after a peripheral nerve injury asdetected by isotopic in situ hybridization.

FIGS. 4D-4F are a series of graphs representing GTPCH, DHPR, andsepiapterin reductase (SPR) mRNA levels as measured by Northern Blotanalysis in the DRG over a period of 14 days following peripheral nerveinjury (*p<0.05; **p<0.01; ***p<0001).

FIGS. 5A and 5B represent a series of graphs showing protein levels andenzyme activity of GTPCH before and following peripheral nerve injury(*p<0.05; **p<0.01; ***p<0001).

FIG. 5C is a bar graph showing BH4 levels in the DRG before andfollowing peripheral nerve injury.

FIG. 6A are a series of graphs showing the ratio of experimental tonaive intensity readings from triplicate Affymetrix microarrays overtime, of the various members of the BH4 synthetic pathway in threedifferent neuropathic pain models (spared nerve injury (SNI), chronicconstriction injury (CCI) and the spinal nerve ligation (SNL) models.FIG. 6A shows the expression profile in the DRG. Changes in theexpression of BH4 synthetic enzymes in the dorsal horn are shown in FIG.6B. The enzymes are present both in the DRG and dorsal horn.

FIGS. 7A and 7B are a series of graphs showing the ratio of experimentalto naive intensity readings from triplicate Affymetrix microarrays overtime of the various members of the BH4 synthetic pathway in the DRG (7A)and dorsal horn (7B) after peripheral inflammation produced byintraplantar complete Freund's adjuvant.

FIGS. 8A-8D represent a series of graphs showing the effect of DAHP onnociceptive behavior in the SNI model of neuropathic pain. Figures onthe left show the threshold to mechanical stimuli applied with von Freyhairs (von Frey threshold). Figures on the right show the duration ofpaw licking, shaking and lifting following acetone application to thepaw as a measure for cold allodynia. In FIG. 8A, rats were treated withDAHP (180 mg/kg/d i.p.) for three days starting three days after surgery(early treatment). Nociceptive behavior was assessed daily before andafter drug injection. In FIG. 8B, the treatment period was extended tofive days (180 mg/kg/d i.p.) starting three days after surgery and painbehavior was assessed daily before and after drug injection and onceevery second day for an additional week after stopping the daily druginjections. In FIG. 8C, treatment with DAHP (180 mg/kg/d i.p. for 5days) was started 17 days after surgery i.e. in the chronic phasewithout any treatment in the early phase. Nociceptive behavior wasassessed once daily 3h after drug injection. In FIG. 8D, animalsreceived a continuous spinal infusion of DAHP (6 mg/kg/d) for 14 days.The infusion was started directly after surgery. Nociceptive behaviorwas assessed once daily.

FIGS. 9A and 9B are two graphs showing the effect of DAHP on nociceptivebehavior in the CCI model of neuropathic pain. FIG. 9A shows thethreshold to mechanical stimuli applied with von Frey hairs (von Freythreshold). FIG. 9B shows the duration of paw licking, shaking andlifting following acetone application to the paw as a measure for coldallodynia. DAHP treatment (180 mg/kg/d i.p. for 5 days) was started 3days after surgery and nociceptive behavior was assessed daily beforeand after drug injection and once every second day after stopping dailydrug injections.

FIG. 10 shows the effect of DAHP on the flinching behavior in theFormalin test. DAHP (180 mg/kg) was injected i.p. one hour beforeinjection of formalin into the hindpaw. Flinches were counted for onehour starting right after formalin injection.

FIGS. 11A-11E are a series of graphs showing the effect of DHAP onthermal hyperalgesia in the CFA model which is a model for inflammatorypain. FIG. 11A shows the paw withdrawal latency in response to radiantheat (Hargreaves test) of the CFA treated, inflamed paw. On the left endof the graph, two doses of DAHP (180 mg/kg i.p.) were injected, thefirst 30 minutes before injection of CFA into the hindpaw, the second 4hours after CFA (indicated with arrows D1 and D2). On the right, asingle dose (dotted arrow D1) of DAHP (180 mg/kg i.p.) was injected 24 hafter induction of paw inflammation without any treatment during thefirst 24 h following CFA injection. FIG. 11B shows the respective pawwithdrawal latencies of the non-inflamed contralateral paw. In FIGS.11C-11D, the effects of systemically administered DAHP (180 mg/kg i.p.single dose; 11C) are compared with the effects of a single intrathecaldose (1 mg/kg i.t.; 11D) which was administered via a lumbar spinalcatheter. In both cases, treatment was started 24 hours after CFAinjection into the hindpaw. Because of slightly different baselinelevels (11C compared with 11D) effects of DAHP after i.p. and i.t.treatment are additionally presented as percentage change of pawwithdrawal latency (FIG. 11E) to allow for a direct comparison of bothroutes of administration.

FIG. 12 is a bar graph showing the increase of the paw weight of theinflamed paw compared with the contralateral paw. Paw weight wasdetermined 48 hours following CFA injection. The increase of the pawweight is a measure for the inflammatory paw edema and therefore allowsassessment of paw inflammation.

FIGS. 13A and 13B are a series of graphs showing the effects of DAHP onthe response to tactile (13A) and heat stimuli (13B) in naïve rats. Asingle dose of DAHP (180 mg/kg i.p.) or vehicle was injected at time“zero”.

FIGS. 14A-14C represent a series of graphs showing pharmacokineticfeatures of DAHP and the pharmacokinetic-pharmacodynamic relationship.FIG. 14A shows the time course of DAHP plasma concentrations followingi.p. injection of a single dose of 180 mg/kg at time “zero”. Inaddition, concentrations of DAHP in cerebrospinal fluid (CSF) weredetermined at an early and late time point. In FIG. 14B, plasmaconcentrations were fitted according to a one-compartment PK model withfirst order input and first order elimination. PK parameters arepresented where Cmax is the maximum concentration, tmax the time of themaximum concentration, k01 is the absorption rate constant, t1/2abs.represents the absorption half-life, k10 is the elimination rateconstant, t1/2 e1 is the elimination half-life, and C1 is the clearance.In FIG. 14C, pooled plasma concentration and effect data of the CFAmodel were used to assess the PK/PD relationship employing a standardsigmoidal Emax model.

FIGS. 15A and 15B show the effect of the sepiapterin reductaseinhibitor, N-acetyl-serotonin (NAS) on thermal hyperalgesia in the CFAmodel. The paw withdrawal latency (PWL) to radiant heat (Hargreavestest) is a measure for the heat sensitivity. NAS treatment (single doseof 50 mg/kg i.p.) was started 24 hours after CFA injection into thehindpaw. Data are presented as percentage change of the PWL comparedwith the PWL of the non-inflamed contralateral paw. FIG. 15B shows theincrease of the paw weight of the inflamed paw compared with thecontralateral paw. Paw weight was determined 48 hours following CFAinjection. The increase of the paw weight is a measure for theinflammatory paw edema.

FIGS. 16A-16D show the effects of systemic (16A) and intrathecal (16B)treatment with methotrexate (MTX) on the nociceptive behavior in the SNImodel of neuropathic pain. In FIGS. 16A, and 16B the left panel showsthe threshold to mechanical stimuli applied with von Frey hairs (vonFrey threshold). The right panel shows the duration of paw licking,shaking and lifting following acetone application to the paw as ameasure for cold allodynia. In FIG. 16A MTX (0.2 mg/kg/d) was injectedi.p. once daily starting 5 days after SNI surgery. Nociceptive behaviorwas assessed once daily three hours after drug injection. In FIG. 16B,MTX was administered as continuous intrathecal infusion (0.1 mg/kg/d for14 days) starting right after SNI surgery. Nociceptive behavior wasassessed once daily. FIGS. 16C and 16D show the body weight gain ofanimals during systemic (16C) and intrathecal (16D) MTX treatment.Determination of the weight gain was used to assess general well-beingand potential toxic effects of MTX.

FIG. 17 is a table showing various sepiapterin reductase inhibitors(Smith et al., (1992) J Biol Chem 267: 5599-5607).

FIG. 18 shows the chemical structures of BH4, guanine, and DAHP

FIG. 19 is a table representing potential GTPCH inhibitors.

FIG. 20 shows the structure of GTPCH and the binding of its substrate tothe catalytic center of the enzyme. Hydrogen bonds between aminoacids ofGTPCH and the substrate GTP are shown as dotted lines.

FIG. 21 shows the interaction of GTPCH-I with the feedback regulatoryprotein (GFRP) and binding site of phenylalanine in the interface ofboth proteins. The binding site of BH4 and GFRP-dependent GTPCH-Iinhibitors is thought to be similar to that of phenylalanine.

FIGS. 22A and 22B are a series of graphs demonstrating that theanti-nociceptive effect of DAHP follows a dose-response relationship formechanical stimuli (FIG. 22A) and thermal stimuli (FIG. 22B) in the SNImodel of nerve injury. In this graph, closed triangles representvehicle, i.p.; open triangles represent DAHP at 90 mg/kg/day, i.p.; opensquares represent DAHP at 180 mg/kg/day, i.p.; and open circlesrepresent DAHP at 270 mg/kg/day, i.p.

FIGS. 23A and 23B are a series of graphs demonstrating thepro-nociceptive effects of BH4. FIG. 23A demonstrates that intrathecaladministration of BH4 reduces the paw withdrawal latency to a thermal(heat) stimulus in naive rats. FIG. 23B demonstrates the pro-nociceptiveeffects of BH4 in animals having a pre-existing thermal hypersensitivity(ipsilateral) compared to controls (contralateral). Hypersensitivity wasinduced using the CFA model of paw inflammation. In these experiments,BH4 was injected at time “zero” after measurement of baseline pawwithdrawal latency.

FIG. 24A is a series of photo micrographs showing cFos immunoreactivityin ipsilateral dorsal horn neurons two hours after formalin injection inDAHP and vehicle-treated rats. FIG. 24B is a bar graph quantifying thenumber of cFos immunoreactive cell bodies observed under each condition.

FIG. 25 is a bar graph showing the number of apoptotic cell profiles,using TUNEL staining, observed in the L4/L5 dorsal horn 7 days after SNIsurgery of animals treated with either 180 mg/kg/day DAHP or vehiclecontrol. Apoptotic neurons were detected by in situ TUNEL labeling andcounted by a blinded observer.

FIG. 26A is a series of graphs demonstrating that there is nosignificant difference between wild-type and nNOS knockout mice, usingthe SNI model either with or without treatment using DAHP, in responseto mechanical or thermal (cold) stimuli. In this figure, closed circlesrepresent nNOS knockout mice with DAHP; open circles, nNOS knock outmice with vehicle; closed triangles, wild type mice with DAHP; and opentriangles, wild type mice with vehicle. FIG. 26B is a series of linegraphs demonstrating that L-NAME, a NOS inhibitor, does not enhance theanti-nociceptive effects of DAHP to mechanical or thermal (cold) stimuliin the SNI model. In this figure, closed triangles represent L-NAMEadministration; open circles, L-NAME+DAHP administration; closedcircles, DAHP administration; and open triangles, vehicleadministration. L-NAME was administered at 25 mg/kg, i.p., single dose,and DAHP was administered at 120 mg/kg, i.p., single dose. Theantinociceptive efficacy of this high L-NAME dose is weaker (about 50%)than that of a moderate dose of DAHP.

FIG. 27A is a series of photomicrographs of in situ hybridization ofGTPCH-I in the ipsilateral (lesioned) and contralateral (control) dorsalroot ganglia 3 days (top panel) and 14 days (bottom panel) after SNIsurgery. FIG. 27B is a series of photomicrographs of GTPCH-I in situhybridization 14 days after SNI surgery and treatment with either DAHPor vehicle control. These data demonstrate that DAHP does not affectGTPCH-1 expression in the SNI model.

FIG. 28A is a series of photomicrographs demonstrating that GTPCH-1 mRNAis not normally expressed in the spinal cord but, three days after SNIsurgery, isolated GTPCH-1-expressing motor neurons could be observed.FIG. 28B is a series of photomicrographs demonstrating that GFRP isexpressed in isolated DRG neurons, but that the expression pattern doesnot change three days after SNI surgery.

FIG. 29 is a series of photomicrographs showing, by in situhybridization, the expression of GTPCH-I (left column) and, byimmunofluorescence, the expression of NF200, Griffonia simplicifoliaisolectin B4 (IB4), CGRP, and nNOS (center column). The number of cellsco-expressing GTPCH-I and each of the identified proteins is expressedas a percentage of cells expressing GTPCH-I (right column).

FIG. 30 is a series of bar graphs quantifying the levels of the BH4metabolites biopterin and neopterin in the DH, DRG, and ScN followingSNI surgery with and without DAHP treatment, compared to control.

FIG. 31 are photomicrographs showing the co-localization of GTPCH-ImRNA, using in situ hybridization, and ATF-3 protein localization, usingimmunohistochemistry, following the SNI neuronal injury model. Thearrows indicate cells that express GTPCH mRNA expression and alsonuclear staining for ATF-3. Overall, 80-90% of GTPCH expressing neuronswere ATF-3 positive demonstrating that GTPCH-1 upregulation occursmostly in injured neurons.

DETAILED DESCRIPTION

In general, the present invention features methods and compounds fortreating, reducing, or preventing pain or a traumatic, metabolic ortoxic peripheral nerve lesion by administering to a mammal atherapeutically effective amount of a composition that reduces the levelor activity of tetrahydrobiopterin (BH4). The invention also providesmethods for diagnosing pain or a traumatic, metabolic or toxicperipheral nerve lesion in a mammal by detecting an increase in thelevels of BH4, BH4 intermediates, BH4 precursors, or BH4 metabolites ina biological sample obtained from a mammal (e.g., serum, plasma, urine,cerebrospinal fluid, synovial fluid, tissue exudates, or tissuesamples). Alternatively, pain may be diagnosed by detecting an increasein the activity or levels of any one of the BH4 synthetic enzymes. Inyet another general embodiment, the invention provides methods foridentifying novel therapeutics for pain based on their ability todecrease the level or activity of BH4 or its synthetic enzymes or itsability to interfere with binding of BH4 to a BH4 receptor orBH4-dependent enzyme.

The invention stems from our discovery that inhibiting the synthesis ofBH4 by interfering with the biological activity of any of the enzymesinvolved in the synthesis of BH4 (e.g., GTP cyclohydrolase (GTPCH),Sepiapterin Reductase (SPR), and Dihydropteridine reductase (DHPR), seeFIG. 2A) results in prevention, treatment, and reduction in pain, i.e.analgesia. To this end, we show that the administration of 2,4 diamino6-hydroxypyrimidine (DAHP), an inhibitor of GTPCH; Methotrexate (MTX),an inhibitor of DHPR; or N-acetyl-serotonin (NAS), an inhibitor of SPR,results in profound analgesia in various rat models of acute post-injurypain hypersensitivity, peripheral neuropathic and inflammatory pain, andwithout an effect on basal pain sensitivity, sedative effect, or grossdisruption of motor function. This reduction in pain is mediated, atleast in part, by the interference with or by a reduction in theactivity of various enzymes for which BH4 is a key co-factor. Suchenzymes include, for example, nitric oxide synthases and catecholaminesynthetic enzymes. In addition, the reduction in the levels of BH4 mayalso reduce its release from cells in the nervous system and its directactions, for example on neurotransmitter release or ion channel activitywhich may be mediated through BH4 binding to membrane bound receptorsthat modify neuronal function.

The methods of this invention are therefore useful for the diagnosis,treatment, reduction, or prevention of various forms of clinical pain,namely inflammatory pain, functional pain and neuropathic pain, whetheracute or chronic. Exemplary conditions that may be associated with paininclude, for example, soft tissue, joint, bone inflammation and/ordamage (e.g., acute trauma, osteoarthritis, or rheumatoid arthritis),myofascial pain syndromes (fibromylagia), headaches (including clusterheadache, migraine and tension type headache), myocardial infarction,angina, ischemic cardiovascular disease, post-stroke pain, sickle cellanemia, peripheral vascular occlusive disease, cancer, inflammatoryconditions of the skin or joints, diabetic neuropathy, and acute tissuedamage from surgery or traumatic injury (e.g., burns, lacerations, orfractures). The present invention is also useful for the treatment,reduction, or prevention of musculo-skeletal pain (after trauma,infections, and exercise), neuropathic pain caused by spinal cordinjury, tumors, compression, inflammation, dental pain, episiotomy pain,deep and visceral pain (e.g., heart pain, bladder pain, or pelvic organpain), muscle pain, eye pain, orofacial pain (e.g., odontalgia,trigeminal neuralgia, glossopharyngeal neuralgia), abdominal pain,gynecological pain (e.g., dysmenorrhea and labor pain), pain associatedwith nerve and root damage due to trauma, compression, inflammation,toxic chemicals, metabolic disorders, hereditary conditions, infections,vasculitis and autoimmune diseases, central nervous system pain, such aspain due to spinal cord or brain stem damage, cerebrovascular accidents,tumors, infections, demyelinating diseases including multiple sclerosis,low back pain, sciatica, and post-operative pain. Conditions that areamenable to treatment according to the present invention are describedin detail, for example, in U.S. Ser. No. 10/348,381 as well as U.S. Pat.Nos. 6,593,331 and 6,593,331, all of which are hereby incorporated byreference.

Briefly, we conducted a cluster analysis of changes in expression ofdevelopmentally- and peripheral nerve injury-regulated genes in thedorsal root ganglion using high-density oligonucleotide microarrays.Interestingly, we found that a particular cluster of genes wascharacterized by high levels of expression during development,down-regulation during adulthood, and induction following nerve injury.Such exemplary genes include DHPR (a member of the BH4 syntheticpathway).

A triplicate analysis of lumbar DRG microarrays three days post-axotomy(sciatic nerve transection) further revealed that three of theapproximately 200 genes that were up-regulated were members of the BH4synthetic pathway (e.g., GTPCH, SPR and DHPR). Their induction undersuch conditions was validated by Northern blot analysis, Northern slotblots, in situ hybridization, and Western blot analysis. We furtherconfirmed that three of the four members of the BH4 synthetic pathwaywere up-regulated in primary sensory neurons after peripheral nerveinjury in at least three models of peripheral neuropathic pain and thatthis was concomitant with an increase in BH4 levels in the dorsal rootganglion. Furthermore, using DAHP (a GTPCH inhibitor) as well as variousother inhibitors of BH4 synthetic enzymes (e.g., NAS and methotrexate),we showed that inhibition of the BH4 synthetic pathway induces analgesiain numerous neuropathic pain models as well as in inflammatory pain anda post-injury pain hypersensitivity model. The analgesic action could bedemonstrated after systemic delivery and intrathecal delivery. Thelatter indicates an action on the nervous system including the DRG andspinal cord. The degree of analgesia matched, or in the case of thespared nerve injury peripheral neuropathic pain model, was far greaterthan that achieved by any of the conventionally used analgesics,including morphine, gabapentin, carbamazepine, amytryptiline, androfecoxib.

Therapeutic Agents Inhibitors of the BH4 Pathway

BH4 is enzymatically synthesized de novo from guanosine 5′-triphosphate(GTP) via two intermediates, 7,8-dihydroneopterin triphosphate and6-pyruvoyl tetrahydropterin (see FIG. 2A). According to the presentinvention, the administration of any agent that inhibits or modulatesthe biological activity of at least one, two, three, or more than threeof any of the BH4 synthetic enzymes shown in FIG. 2A to reduce BH4synthesis induces analgesia. Such enzymes include, for example,sepiapeterin reductase (SPR), Pyruvoyltetrahydropterin Synthase (PTPS),GTP cyclohydrolase (GTPCH), Pterin-4α-carbinolamine dehydratase, anddihydropteridine reductase (DHPR). The analgesic effect that resultsfrom the reduction in BH4 levels is caused, at least in part, by areduction in the biological activity of enzymes for which BH4 is aco-factor and of membrane-bound receptors to which BH4 binds.

In this regard, BH4 is an essential cofactor of several enzymes (e.g.,the hydroxylases of the three aromatic amino acids phenylalanine,tyrosine, and tryptophan;

ether lipid oxidase; and the three nitric oxide synthase (NOS)isoenzymes, eNOS, iNOS, and nNOS) and therefore plays a key role in anumber of biological processes, including neurotransmitter formation andsignaling in pain pathways. Indeed, a number of enzymes that areregulated by BH4 have previously been involved in pain and include forexample, NOS (Meller et al. (1992) Neuroscience 50:7-10; Minami et al.,(1995) Neurosci. Lett. 201:239-242; Yamaguchi and Naito (1996) Can. J.Anaesth. 43:975-981; Aley et al., (1998) J. Neurosci. 18:7008-7014;Handy and Moore (1998) Neuropharmacology 37:37-43; Levy and Zochodne(1998) Eur. J. Neurosci. 10:1846-1855; Guhring et al. (2000) J.Neurosci. 20:6714-6720; Levy et al. (2000) Eur J Neurosci 12:2323-2332,tyrosine hydroxylase (Ma and Bisby (1999) Neurosci Lett 275:117-120;Lindqvist et al. (2000) Muscle Nerve 23:1214-1218) and tryptophanhydroxylase.

According to the present invention, an inhibitor of the BH4 pathway isany agent having the ability to reduce the production or the activity ofBH4 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%relative to an untreated control cell as determined by any standardmethod in the art, including those described herein. Alternatively, theinhibitor may treat, prevent, or reduce pain when administered to amammal by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%relative to an untreated control. Such reduction or prevention in painmay be measured by any technique known in the art such as thosedescribed herein. Exemplary compounds that may be used according to thisinvention include DAHP, an inhibitor of GTPCH; MTX, an inhibitor ofDHPR; NAS, an inhibitor of SPR; Tetrahydro-L-biopterin, L-Sepiapterin,7,8-dihydro-L-Biopterin, 6,7-dimethyltetrahydropterin hydrochloride,N-acetyl-serotonin (NAS), N-Chloroacetylserotonin,N-Methoxyacetylserotonin, N-Chloroacetyldopamine, as well as any of thecompounds identified by any of the screening methods of the invention.Other agents that may be used are described further below.

Optionally, the BH4 inhibitor may be a small molecule antagonist or anantisense to any of the BH4 synthetic enzymes. RNA interference (RNAi)may also be used to target the BH4 synthetic pathway as it provides apowerful method of gene silencing in eukaryotic cells includingmammalian cells such as the primary sensory neurons of the presentinvention. The basic technique of RNAi involves introducingsequence-specific double-stranded RNA into neurons in order to generatea nonheritable, epigenetic knockout of gene function that phenocopies anull mutation in the targeted gene. RNA interference has previously beendescribed (O'Neil N J, et al., Am J Pharmacogenomics (2001): 45-53).

Alternatively, the analgesic agent may be a dominant negative protein ora nucleic acid encoding a dominant negative protein that interferes withthe biological activity of BH4 or any of the BH4 synthetic enzymes. Adominant negative protein is any amino acid molecule having a sequencethat has at least 50%, 70%, 80%, 90%, 95%, or even 99% sequence identityto at least 10, 20, 35, 50, 100, or more than 150 amino acids of thewild type protein to which the dominant negative protein corresponds.For example, a dominant-negative BH4 synthetic enzyme may have mutationsuch that it no longer able to produce BH4.

According to this invention, the dominant negative protein may beadministered as an expression vector. The expression vector may be anon-viral vector or a viral vector (e.g., retrovirus, recombinantadeno-associated virus, or a recombinant adenoviral vector).Alternatively, the dominant negative protein may be directlyadministered as a recombinant protein to dorsal root ganglia or thespinal cord using, for example, microinjection techniques.

Inhibitors of GTP Cyclohydrolase I (GTPCH)

The rate-limiting enzyme in BH4 de novo biosynthesis is GTPcyclohydrolase I (GTPCH), which converts GTP to 7,8-dihydroneopterintriphosphate (see FIG. 2A). Over time, the accumulation of BH4 causes afeedback inhibition of GTPCH, in a reaction mediated by the GTPCHFeedback Regulatory Protein (GFRP). In the presence of phenylalanine,GFRP induces a feed-forward activation of GTPCH activity by enhancingGTP binding (Maita et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99:1212-1217). However, in the presence of BH4, GFRP induces feedbackinhibition of GTPCH enzyme activity (Yoneyama and Hatakeyama, (1998) J.Biol. Chem. 273:20102:20108); Yoneyama and Hatakeyama, (2001) ProteinSci. 10: 981-878) such that BH4 production is auto-inhibited.

Accordingly, any agent that inhibits GTPCH activity can efficientlyreduce the production of BH4 and in turn, induce analgesia. GTPCHinhibitors may compete with the substrate GTP for binding to thecatalytic center of the enzyme (FIG. 20). Alternatively, GTPCHinhibitors (for example BH4 analogs) may inhibit BH4 production bybinding to the GTPCH/GFRP complex (FIG. 21) and thereby mimic thefeedback inhibition of BH4. Although GFRP mRNA is abundant in brainstemneurons, it remains undetectable in dopamine neurons of the midbrain andin norepinephrine neurons of the locus coeruleus (Kapatos et al., (1999)J. Neurochem. 72: 669-675). Thus, the GFRP-dependency of GTPCHinhibitors may confer specificity and simultaneously avoiddopamine-related side effects.

Here, we have shown, for example, that the specific GTPCH inhibitor,2,4-Diamino-6-hydroxypyrimidine (DAHP), induces analgesia in variousneuropathic pain and inflammatory models (see FIGS. 8-13). DAHP-mediatedinhibition of GTPCH is mediated by GFRP-dependent (at lowconcentrations) and GFRP-independent (at higher concentrations)mechanisms. Accordingly, DAHP mimics BH4 in its indirect mechanism ofGTPCH inhibition at low concentrations. At higher concentrations DAHPcompetes with the physiological GTPCH-substrate, guanosine-triphosphate(GTP) for binding to the catalytic site (Xie et al., (1998) J. Biol.Chem. 273:21091-21098).

Biochemical and crystallographic studies on the interaction of GTPCHwith GTP reveal that hydrogen bonds are formed between highly conservedamino acids found within the active site of GTPCH and the pyrimidineportion of guanine i.e. the nitrogen atoms at position 1, 2 and 3 andthe oxygen at position 6, respectively (see FIG. 20) (Rebelo et al.,(2003) J. Mol. Biol. 326: 503-516). This particular portion of theguanine structure exactly matches the pyrimidine structure of DAHP aswell as a portion of the pteridine structure of tetrahydrobiopterin(BH4), which is the end product of the enzymatic cascade.

The structural homology between DAHP, guanine, and BH4 (see FIG. 18)indicates that it is the pyrimidine portion in these molecules that islikely to be crucial for binding to GTPCH. Analysis of variouspyrimidine-molecules with modifications at these sites reveals thatalterations at position 2, 4 and 6 are associated with a reduction orloss of GTPCH inhibitory activity (Yoneyama et al. (2001) Arch. Biochem.Biophys. 388:67-73) (see FIG. 19). On the other hand, guanine itself(i.e. part of the physiological substrate) and guanine analogs withmodifications at position 7 or 8 inhibit GTPCH with a higher potency(approximately 10 fold higher than DAHP) (Yoneyama et al., supra) (FIG.19). Thus, drugs containing a 5-membered ring like guanine (thus alsosharing higher similarity with BH4) have an increased inhibitory effect.Interestingly, BH4 is about 10 times more potent than guanine analogs(Yoneyama et al., supra) (FIG. 19) and 100 times more potent than DAHP,therefore suggesting that the side chain attached to C-6 of BH4 confersfurther potency and/or specificity. This is supported by the findingthat an 6S-BH4 enantiomer and an analog of BH4 with no side chain at C-6are more than one order of magnitude less effective than BH4 (Harada etal. (1993) Science 260:1507-1510). Accordingly, the potency of DAHP canbe increased, for example, by increasing its structural similarity toBH4 while retaining substitutions or modifications that prevent its useas a cofactor for BH4-dependent enzymes (e.g., tyrosine, phenylalanine,and tryptophan hydroxylases; glycerol ether monooxygenases; and nitricoxide synthases (which utilize various N-alkyl andN-aryl-hydroxyguanidines such as N-(4-Chlorophenyl)N′-hydroxyguanidineas substrates for the production of nitric oxide (Moali et al. (2001)Chem. Res. Toxicol. 14:202-210; Renodon-Corniere et al., (1999)Biochemistry 38: 4663-8)).

In light of the above, the mammal being treated according to the presentinvention may be administered with a composition containing a compoundhaving the formula:

such that R¹ is a H, C₁₋₆ alkyl, halo, NO₂, CN, CO₂R⁴, CONR⁴R⁵, SO₂R⁴,SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵. Each of R⁴ and R⁵ may be, independently, a H,C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl;R² may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl; and R³ may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶, CONR⁷R⁸, SO₂R⁶, or SO₂NR⁷R⁸,where R⁶ is a C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl, and each of R⁷ and R⁸ is, independently, a H, C₁₋₆ alkyl,C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl.

Alternatively, R³ may be as above and R¹ and R² together may berepresented by

where the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Optionally, R³ may be as above and R¹ and R² together may be representedby

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² or R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, such that each of R⁷ and R⁸ is,independently, a H, C₁-C₆ alkyl, C₆-C₁₂ aryl, heteroaryl, C₁-C₁ alkaryl,or C₁-C₁ alkheteroaryl.

As another alternative, R¹ and R² together may be represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing the R¹⁴ and thenitrogen bearing R².

Optionally, the composition may have be a compound of formula (I), suchthat R¹ is a H, C₁₋₆ alkyl, halo, NO₂, CN, CO₂R⁴, CONR⁴R⁵, SO₂R⁴,SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵. Accordingly, each of R⁴ and R⁵ may be,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl, R² may be a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, and R³ may be a H, C₁₋₆ alkyl,C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶,CONR⁷R⁸, SO₂R⁶, or SO₂NR⁷R⁸, where R⁶ is a C₁₋₆ alkyl, C₆₋₁₂ aryl,heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl and each of R⁷ and R⁸may be, independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl.

If desired, the composition contains a compound of formula:

in which R¹ and R² together may be represented by

such that the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R³ is as above and R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² and R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, where each of R⁴, R⁷ and R⁸ is,independently, a H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R¹ and R² together may be represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².

If the composition contains a compound of formula:

R¹ and R² together may be represented by

where the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Alternatively, the composition contains a compound of formula:

in which R³ is as above and R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R¹² and R¹³ is as above, and R¹⁴ is a OR⁴, halo, NO₂, CN, CO₂R⁷,CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, such that each of R⁷ and R⁸ is,independently, H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl.

Optionally, the composition may contain a compound of formula:

in which R¹ and R² together are represented by

where the N of the R¹/R² linkage forms a bond to the pyrimidinone ring,each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, and adouble bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².

Exemplary compounds that may be contained within the composition of theinvention include, for example:

These compounds are shown in FIG. 19.

Furthermore, the composition of the invention may contain a compoundhaving the formula:

in which R¹⁵ is a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl,or C₁₋₄ alkheteroaryl and R¹⁶ is a H, C₁₋₆ alkyl, C₆₋₁₂ aryl,heteroaryl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R¹⁷, CONR¹⁸R¹⁹, SO₂R¹⁷,or SO₂NR¹⁸R¹⁹. R¹⁷ may be a C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl and each of R¹⁸ and R¹⁹ may be,independently, a H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.

Further exemplary compounds include Tetrahydro-L-biopterin (BH4.2HCl, areduced pterin that is a noncompetitive inhibitor of GTP cyclohydrolaseI with a Ki of 15.7 μM); L-Sepiapterin (a reduced pterin that is 12times more potent than oxidized pterins as a GTP cyclohydrolase Iinhibitor and has an IC50 of 12.7±1.8 μM); 7,8-dihydro-L-Biopterin (BH2,a metabolic end product of GTP cyclohydrolase I in vitro, whichfunctions as a noncompetitive inhibitor of GTP cyclohydrolase I (Ki of14.4 μM) and is approximately 12 times more potent as an inhibitor thanoxidized pterins, folates, and aminopterins); and6,7-dimethyltetrahydropterin hydrochloride (a noncompetitive inhibitorof GTP cyclohydrolase I (IC50 of 76 to 112 μM)). As discussed above, theinhibition of GTPCH may also be accomplished using inhibitors that acton GTPCH in a GFRP-independent GTP competitive fashion (e.g., guaninederivatives as shown in FIG. 19 and as described above).

Activators of GTPCH Feedback Regulatory Protein (GFRP)

GFRP is the endogenous inhibitor of BH4 synthesis by GTPCH. The bindingof GFRP to GTPCH is, itself, a BH4-dependent event, making GFRP anegative feedback regulator of BH4 production. It has recently beendiscovered that DAHP, a molecule that is structurally similar to BH4,binds to GFRP and promotes GTPCH inhibition (Kolinsky et al., J. Biol.Chem., Manuscript M405370200, Jul. 29, 2004). Thus, molecules that mimicthe actions of BH4 and/or DAHP at GFRP by enhancing the binding of GFRPto GTPCH and inhibiting the production of BH4 are useful in the methodsof this invention. Alternatively, molecules that facilitate the bindingof GFRP and GTPCH, but not through a binding at the BH4 site on GFRP arealso useful. Such molecules include, for example, bi-functionalantibodies or cross-linking agents.

Inhibitors of Sepiapterin Reductase

Sepiapterin Reductase (SPR) functions as the final synthetic enzyme inthe BH4 pathway. Here, we show that NAS (50 mg/kg i.p., an inhibitor ofSPR, produces analgesia in a model of inflammatory pain (FIGS. 15A and15B). Other sepiapterin reductase inhibitors with increased potencyinclude N-Chloroacetylserotonin, N-Methoxyacetylserotonin, andN-Chloroacetyldopamine.

Inhibitors of Dihydropteridine Reductase (DHPR)

The BH4 salvage arm, which involves DHPR, allows a recycling of oxidizedBH4 without de novo synthesis. The inhibition of DHPR, which isupregulated in DRGs and the spinal cord following nerve injury, inducesanalgesia by inhibiting the recycling of BH4 from BH2. For example, theadministration of methotrexate (MTX, also known as amethopterin hydrate(0.1 mg/kg/d as continuous intrathecal infusion or 0.2 mg/kg i.p. oncedaily) results in pain reduction without directly affecting BH4 de novosynthesis (see FIGS. 16A-16F). MTX is an inhibitor of dihydrofolatereductase and has been approved for use in humans as animmunosuppressant. It is typically used at low dosages for the treatmentof rheumatoid arthritis (Weinstein et al. (1985) Am J Med 79: 331-7,Williams et al. (1985) Arthritis Rheum;28: 721-30, Weinblatt et al.(1985) N Engl J Med 312: 818-22, Hoffmeister et al. (1983) Am J Med 75:69-73, Giannini et al. (1992) N Engl J Med 326: 1043-9). Whilesystemically administered MTX primarily reaches peripheral targets andDRGs, MTX delivered spinally targets the spinal cord and DRGs.Systemically administered MTX (at low doses) does not penetrate theblood brain barrier because it is a substrate for ATP-binding cassette(ABC) transporters (probenicid-sensitive multi-drug resistance protein(MRP) 1-3). Consequently, systemic treatment with MTX may require theco-administration with probenicid or other inhibitors of MRPs or organicanion transporters to ensure that MTX reaches the spinal cord and brain.

If desired, analgesia may be induced by simultaneously blocking bothparts of the BH4 pathway, namely the biosynthesis and the salvagepathways, to achieve an additive effect. For example, the reduction inpain may be significantly enhanced by blocking BH4 synthesis bysimultaneously inhibiting GTPCH using DAHP and DHPR using methotrexate(or other DHPR inhibitors that achieve higher concentration in thecentral nervous system).

Inhibitors of Pterin-4α-carbinolamine dehydratase (PCD)

Quinoid dihydropterin products are strong inhibitors of thePterin-4α-carbinolamine dehydratase (PCD) (having KI's of about one halfof their respective Km's) and may therefore be used according to thepresent invention. (Rebrin et al. (1995) Biochemistry 34: 5801-10).

Second Therapeutic Agents

The composition of the present invention may be administered eitheralone or in combination with a second therapeutic agent, such as ananalgesic agent used in the treatment of nociception, inflammatory,functional or neuropathic pain. According to this invention, the secondtherapeutic agent may or may not produce a therapeutic effect whenadministered on its own, but results in such an effect (e.g., painreduction) when administered with the composition of the invention.

Exemplary analgesic agents include, nonsteroidal anti-inflammatoryagents (NSAIDs) (e.g. rofexocib, celecoxib, valdecoxib, paracoxib,salicylic acid, acetominophen, diclofenac, piroxican indomethacin,ibuprofen, and naproxen), opioid analgesics (e.g., propoxyphene,meperidine, hydromorphone, hydrocodone, oxycodone, morphine, codeine,and tramodol), NMDA antagonist analgesics (e.g., 2-piperdino-1 alkanolderivatives, ketamine, dextormethorphan, eliprodil, or ifenprodil),anesthetic agents (e.g., nitrous oxide, halothane, fluothane) localanesthetics (lidocaine, etidocaine, ropivacaine, chloroprocaine,sarapin, and bupivacaine), benzodiazepines (diazepam, chlordiazepoxide,alprazolam, and lorazepam), capsaicin, tricyclic antidepressants (e.g.,amitriptyline, perphanazine, protriptyline, tranylcypromine, imipramine,desimipramine, and clomipramine), skeletal muscle relaxant analgesics(flexeril, carisoprodol, robaxisal, norgesic, and dantrium), migrainetherapeutic agents (e.g., elitriptan, sumatriptan, rizatriptan,zolmitriptan, and naratriptan), anticonvulsants (e.g., phenytoin,lamotrigine, pregabalin, carbamazepine, oxcarbazepine, topiramate,valproic acid, and gabapentin), baclofen, clonidine, mexilitene,diphenyl-hydramine, hydroxysine, caffeine, prednisone, methylprednisone,decadron, paroxetine, sertraline, fluoxetine, tramodol, ziconotide,levodopa.

If desired, the mammal being treated may be administered with more thanone agent that inhibits the production of BH4. Optionally, thecomposition of the invention may contain more than one such inhibitor.Alternatively, the mammal may further be administered with specificinhibitors of enzymes that function downstream of BH4, in addition tothe composition of the invention. Such inhibitors are described below.

BH4-depedent Enzymes

BH4 is an essential cofactor of several enzymes, i.e. the hydroxylasesof the three aromatic amino acids phenylalanine, tyrosine, andtryptophan; ether lipid oxidase; and of the three nitric oxide synthase(NOS) isoenzymes. Thus, BH4 plays a key role in a number of biologicalprocesses including neurotransmitter formation and signaling pathways.

Nitric oxide (NO) is released from nociceptive neurons following NMDAreceptor stimulation and diffuses back to the presynaptic neuron whereit causes further glutamate release by stimulating the guanylylcyclase/cGMP/cGMP dependent kinase pathway. Furthermore, NO modulatesexcitability of the postsynaptic neuron. Inhibitors of NO synthase, suchas L-NAME (N^(G)-Nitro-L-arginine-methyl ester), reduce inflammatoryhyperalgesia and neuropathic allodynia in various models andaccordingly, may be administered with or admixed in the composition ofthe invention. Given that drugs, which block the activity of all NOSenzymes, are generally more effective than specific inhibitors of eitherneuronal NOS (nNOS) or inducible NOS (iNOS), both enzymes are mostlikely involved in pain. nNOS is constitutively expressed in neurons andupregulated after peripheral nociceptive stimulation. iNOS isupregulated in the spinal cord after peripheral nociceptive stimulationparticularly in glial cells and produces much higher NO levels thannNOS. Although endothelial nitric oxide synthase (eNOS) is primarilyexpressed in endothelial cells, studies in knockout mice have shown thatthis enzyme also contributes to pain modulation. All of these enzymesare dependent on BH4, employing it as a cofactor. Inhibitors of the NOSpathway that may be used to induce analgesia include inhibitors of NOS-1(nNOS) such as N-Methyl-L-arginine (M 7033), N-Nitro-L-arginine (N5501), 7-Nitroindazole (N 7778), 1-(2-Trifluoromethylphenyl)imidazole (T7313), L-Thiocitrulline, S-Methyl-L-thiocitrulline (M 5171); inhibitorsof NOS-2 (iNOS), such as Aminoguanidine (A 8835, A 7009),S-Benzylisothiourea (B 9138), 1-(2-Trifluoromethylphenyl)imidazole (T7313), L-N6-(1-Iminoethyl)lysine (I 8021), and 1400W (W 4262); andinhibitors of NOS-3 (eNOS) include N-Methyl-L-arginine (M 7033),N-Nitro-L-arginine (N 5501), N-Iminoethyl-L-ornithine (I 8768), and7-Nitroindazole (N 7778).

Tyrosine hydroxylase catalyses the first step in cathecholaminesynthesis, i.e., the production of dopamine from the amino acid tyrosinein a reaction that requires the presence of BH4. Tryptophan hydroxylaseis the key enzyme in serotonin synthesis. Noradrenaline and serotoninact as neurotransmitters in descending inhibitory neurons arising fromthe locus coeruleus and nucleus raphe magnus, respectively. Reduction ofco-factor availability for these enzymes may therefore result in anincrease of pain. To overcome this potential disadvantage, thecomposition of the present invention may be administered in combinationwith a 5HT receptor agonist and/or a centrally acting alpha receptoragonist such as clonidine. Various 5HT-1 agonists have antinociceptiveeffects when administered alone such as the 5-HT-1 agonistsm-trifluoromethylphenyl-piperazine (TFMPP) and7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo(1,2-1a)quinoxaline(CGS 12066B) (Alhaider et al., (1993) J Pharmacol Exp Ther. 265:378-85)or the 5HT-2A agonist, FR143166 (Ochi et al. (2002) Eur J Pharmacol.452:319-24). Alternatively, a potential decrease of serotonin and/ornoradrenaline synthesis may be outweighed by combination treatment witha reuptake inhibitor such as a tricyclic antidepressant (e.g.amitriptyline) or a selective serotonin reuptake inhibitor (e.g.paroxetine, see above).

Direct Effects of BH4

In addition to its co-factor function, the R enantiomer of BH4 (6R-BH4)exhibits various direct effects on neurons when delivered viamicrodialysis to certain regions of the brain. For example, 6R-BH4increases the release of dopamine in the striatum. Given that theseeffects persist in the presence of a tyrosine hydroxylase inhibitor(alpha-methyl-p-tyrosine), it is probably not caused by an increase ofdopamine synthesis (Koshimura et al. (1995) J Neurochem. 65: 827-30).6R-BH4 also increases the release of other neurotransmitters such asacetylcholine in the hippocampus (Ohue et al. (1991) Neurosci Lett.128:93-6) and glutamate and serotonin in the striatum and frontal cortex(Mataga et al. (1991) Brain Res. 551: 64-71). The neurotransmitterreleasing effects of BH4 are inhibited with a calcium channel antagonist(Koshimura et al. (1995) J Neurochem. 65:827-30). The addition of 6R-BH4to microdialysis perfusion fluid also increases calcium currents in themotor nucleus of the vagus in rats whereas the addition of L-DOPA or thenitric oxide donor Sin-1 has no effect, therefore suggesting that theseeffects are independent of tyrosine hydroxylase or NOS activity,respectively (Shiraki T et al., (1996) Biochem Biophys Res Commun. 221:181-5). The S-enantiomer of BH4 (6S-BH4) or the precursor sepiapterinhas no effect on neurotransmitter release. 6R-BH4 may therefore actthrough a specific “BH4-receptor.” However, such a membrane boundextracellular binding site for BH4 has not yet beenidentified/characterized.

Screening for Potential Novel BH4 Binding Sites

The results described herein suggest the existence of a novel membranebound or intracellular BH4 binding molecule that functions as a BH4target protein. Although the characteristics of BH4 as a coenzyme arewell described in the literature its binding to other proteins and itstransport mechanisms have not been investigated. Novel proteomicapproaches have been developed that allow for a high throughputscreening of binding sites of small molecules such as BH4. Large-scaleprotein chips i.e., two-dimensional displays of individual proteins havebeen constructed by immobilizing large numbers of purified proteins onmicroplates. They are used, for example, to assay protein-proteininteractions, drug-target or enzymes-substrate interactions. Generallythey require an expression library, cloned into E. coli, yeast, or othersimilar expression systems from which the expressed proteins are thenpurified, and immobilized. Any suitable protein purification methodknown in the art may be used including, for example, a His-tag. Cellfree protein transcription/translation such as ribosome display is analternative for synthesis of proteins. Phage or yeast display librariesmay also be used.

Binding of BH4 may be detected directly by labeling BH4 for example withtritium what has been described previously (Werner et al., Biochem. J.604: 189-193, 1994) or with biotin which may be coupled to the primaryamino group of BH4. Incorporation of tritium may be achieved in a cellbased system with over-expression of the synthetic enzymes and[³H]-labeled GTP as the substrate (Werner et al., 1994). BH4 can also bechemically labeled with tritium using the commercially available 6R-BH4hydrochloride as template. As an alternative to BH4 labeling, itsbinding to novel targets might also be detected by using a labeledcapturing molecule e.g., a peptide containing the BH4 binding site ofone of the enzymes that bind BH4 as cofactor or a BH4 antibody.Label-free detection methods, including mass spectrometry, surfaceplasmon resonance and atomic force microscopy are also available andavoid alteration of the ligand. In addition to large scale proteinarrays two-dimensional gel electrophoresis of tissue or cell proteinextracts of the dorsal horn and DRGs can be used to screen for novelbinding sites. The gel is then blotted onto PVDF membranes and exposedto labeled BH4. Positive protein spots can be analyzed by massfingerprinting using matrix-assisted laser desorption ionization time offlight mass spectrometry (MALDI-TOF MS).

Diagnosis of Pain or a Peripheral Nerve Lesion Using the BH4 Pathway

According to this invention, pain or a traumatic, metabolic or toxicperipheral nerve lesion may be diagnosed in a mammal by measuring thelevels of BH4, BH4 intermediates, BH4 precursors, or BH4 metabolites ina biological sample obtained from a mammal (e.g., serum, plasma, urine,cerebrospinal fluid, synovial fluid, tissue exudate, or tissue sample).Pain or a peripheral nerve lesion is diagnosed if an increase in suchlevels relative to a control (at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 100%, or more than 100% relative to control) isdetected using any standard method known in the art. Alternatively, painor a peripheral nerve lesion may be diagnosed if an increase in thelevels or activity of any of the BH4 synthetic enzymes is detected.Desirably, such increase is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 100%, or more than 100% relative to control conditions.According to this invention, the BH4 pathway serves as a biomarker ofnerve injury and pain.

The present invention further provides a kit for diagnosing pain or atraumatic, metabolic or toxic peripheral nerve lesion in mammalinvolving the measurement of BH4, its precursors and intermediates(e.g., 7,8-dihydroneopterin triphosphate neopterin and 6-pyruvoyltetrahydropterin) or metabolites (e.g., pterin, biopterin, 7,8dihydropterin, 7, 8 dihydroxanthopterin, xanthopterin, isoxanthopterin,or leucopterin) in a biological sample, such as serum, plasma, urine,cerebrospinal fluid, synovial fluid, tissue exudates, and tissuesamples. For example, the invention may include an antibody specific forany one of the above compounds (e.g., biopterin or neopterin) andinstructions to diagnose pain in a mammal. In this regard, serum may beisolated from a mammal in which a condition associated with the symptomof pain is tested or a traumatic, metabolic or toxic peripheral nervelesion is suspected, and subjected to an ELISA or RIA assay using anantibody of the invention. Pain is diagnosed in the mammal if the serumlevel of BH4 in the mammal's serum (as detected by the antibody) isincreased relative to the BH4 levels in a control serum sample. Thediagnosis of pain, a subtype of pain, or a peripheral nerve lesion is ina mammal if the levels of the compound being measured in the biologicalsample obtained from the mammal is increased by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to thelevel of the same compound in a control biological sample.

Pain Models

Various models test the sensitivity of normal animals to intense ornoxious stimuli (physiological or nociceptive pain). These tests includeresponses to thermal, mechanical, or chemical stimuli.

Thermal stimuli usually involve the application of hot stimuli(typically varying between 42 -55° C.) including, for example: radiantheat to the tail (the tail flick test), radiant heat to the plantarsurface of the hindpaw (the Hargreaves test), the hotplate test, andimmersion of the hindpaw or tail into hot water. In such models, the endpoints include latency to a painful response, the duration of theresponse, vocalization, and licking of the paw. Immersion in cold water,acetone evaporation, or cold plate tests may also be used to test coldpain responsiveness.

Tests involving mechanical stimuli typically measure the threshold foreliciting a withdrawal reflex of the hindpaw to graded strengthmonofilament von Frey hairs (the outcome measure being the force of thefilament required to elicit a reflex) or to a sustained pressurestimulus to a paw (e.g., the Ugo Basile analgesiometer). The duration ofa response to a standard pinprick may also be measured.

When using a chemical stimulus, the response to the application orinjection of a chemical irritant (e.g., capsaicin, mustard oil,bradykinin, ATP, formalin, acetic acid) to the skin, muscle joints orinternal organs (e.g., bladder or peritoneum) is measured. The outcomemeasures include vocalization, licking of the paw, writhing, orspontaneous flexion.

In addition, various tests assess pain sensitization by measuringchanges in the excitability of the peripheral or central components ofthe pain neural pathway. In this regard, peripheral sensitization (i.e.changes in the threshold and responsiveness of high thresholdnociceptors) can be induced by repeated heat stimuli as well as theapplication or injection of sensitizing chemicals (e.g. prostaglandins,bradykinin, histamine, serotonin, capsaicin, mustard oil). The outcomemeasures are thermal and mechanical sensitivity in the area ofapplication/stimulation using the techniques described above in behavinganimals, or alternatively, electrophysiological measurements of singlesensory fiber receptive field properties either in vivo or usingisolated skin nerve preparations. The electrophysiological,neurochemical or cell biological properties of sensory neurons can alsobe used to study these parameters indirectly (e.g., recordings fromisolated sensory neurons (e.g., dorsal root ganglion neurons inculture), activation of signal transduction pathways by sensitizingstimuli (e.g., protein kinase C or A), or measurements of receptor orion channel phosphorylation). Central sensitization (i.e. changes in theexcitability of neurons in the central nervous system induced byactivity in peripheral pain fibers) can be induced by noxious stimuli(e.g., heat), chemical stimuli (i.e. injection or application ofchemical irritants such as capsaicin, mustard oil, or formalin), orelectrical activation of sensory fibers. The outcome measures may bebehavioral (i.e. thermal and mechanical responsiveness outside of thearea of application, that is the area of secondary hyperalgesia, ortactile allodynia (pain responses to normally innocuous tactilestimuli)), electrophysiological (i.e. receptive field properties ofsingle central neurons), neurochemical (i.e. activation of signaltransduction pathways in central neurons (e.g., ERK, p38, CREB,immediate early genes such as c-fos, kinases, PKC, PKA, or src) orphosphorylation of receptors or ion channels such as NMDA or AMPAreceptors). Functional imaging techniques may also be used to assesschanges in the patterns of activation.

Various pain tests have also been developed to measure the effect ofperipheral inflammation on pain sensitivity (Stein et al., Pharmacol.Biochem. Behay. (1988) 31: 445-451; Woolf et al., Neurosci. (1994) 62:327-331). The inflammation may be produced by injection of an irritant(e.g., complete Freund's adjuvant, carrageenan, turpentine, and crotonoil) into the skin, subcutaneously, into a muscle, into a joint, or intoa visceral organ. Alternatively, the generation of a controlled UV lightburn and ischemia or the administration of cytokines or inflammatorymediators such as lipopolysaccharide (LPS) or nerve growth factor (NGF)can also mimic the effects of inflammation. Following the induction ofinflammation, the outcome measures may include changes in behavior(e.g., thermal and mechanical sensitivity (as discussed above), weightbearing, visceral hypersensitivity (e.g., inflation of balloons inbladder or bowel), spontaneous locomotor activity, or performance inmore complex behaviors such as place preference tasks), inelectrophysiology (e.g., in vivo and in vitro recordings from primarysensory neurons and central neurons with particular attention to changesin receptive field properties, excitability, or synaptic input), inneurochemistry (e.g., changes in the expression and distribution oftransmitters, neuropeptides, and proteins in primary sensory and centralneurons, activation of signal transduction cascades, expression oftranscription factors, and phosphorylation of proteins in neurons), andimaging techniques to detect changes in neural activity.

Additionally, various tests assess peripheral neuropathic pain usinglesions of the peripheral nervous system. One such example is the“axotomy pain model,” for example, which involves the completetransection of a peripheral nerve and in which one or a plurality ofperipheral nerve fibers is severed, either by traumatic injury orexperimental or surgical manipulation (Watson, J. Physiol. (1973)231:41). Other similar tests include the SNL test which involves theligation of a spinal segmental nerve (Kim and Chung Pain (1992) 50:355), the Seltzer model involving partial nerve injury (Seltzer, Pain(1990) 43: 205-18), the spared nerve injury (SNI) model (Decosterd andWoolf, Pain (2000) 87:149), chronic constriction injury (CCI) model(Bennett (1993) Muscle Nerve 16: 1040), tests involving toxicneuropathies such as diabetes (streptozocin model), pyridoxineneuropathy, taxol, vincristine, and other antineoplastic agent-inducedneuropathies, tests involving ischaemia to a nerve, peripheral neuritismodels (e.g., CFA applied peri-neurally), models of post-herpeticneuralgia using HSV infection, and compression models. In all of theabove tests, outcome measures may be assessed, for example, according tobehavior (e.g., thermal and mechanical sensitivity as above, weightbearing, spontaneous activity, or performance in more complex behaviorssuch as place preference tasks), electrophysiology (e.g., in vivo and invitro primary sensory neurons and central neurons with particularattention to changes in membrane excitability, spontaneous activity,receptive field properties, and synaptic input), neurochemistry (e.g.,expression and distribution of transmitters, neuropeptides and proteinsin primary sensory and central neurons, activation of signaltransduction cascades, expression of transcription factors, andphosphorylation of proteins in neurons), and imaging techniques todetect changes in neural activity. Furthermore, several pain tests thatmimic central neuropathic pain involve lesions of the central nervoussystem including, for example, spinal cord injury (e.g., mechanical,compressive, ischemic, infective, or chemical). In these particulartests, outcome measures are the same as those used for peripheralneuropathic pain.

Various features of pain are shared between the above models. In thisrespect, physiological pain is characterized by a high threshold tomechanical and thermal stimuli and rapid transient responses to suchstimuli. Inflammatory and neuropathic pain are characterized by displaysof behavior indicating either spontaneous pain (measured by spontaneousflexion, vocalization, biting, or even self mutilation), abnormalhypersensitivity to normally innocuous stimuli (allodynia), and anexaggerated response to noxious stimuli (hyperalgesia).

Measurement of Pain

Thermal and mechanical threshold sensitivity may be measuredquantitatively, for example, in ° C., force in grams or Newtons, oralternatively, as a measure of time to respond. For thermal painthresholds, the temperature of a hot stimulus >40° C. or a cold stimulus(<15 ° C.) that elicits a flexion withdrawal response is typicallymeasured. For mechanical thresholds the force of a punctate mechanicalstimulus (<100 g) that elicits a flexion withdrawal response ismeasured. The reduction in the latency of response to the stimulus, thatis the length of time the animal takes to respond, can also be measured(typically <10 seconds). The actual values depend on the nature of thetest and the area of the body stimulated. One way of testing an increasein pain sensitivity is to repeatedly apply a stimulus close to thresholdlevels and look for an increase in the proportion of positive responsesto this fixed stimulus. For pain responsiveness, the measurement is theduration or magnitude of a response such as the amount of time an animalholds its limb in a flexed position after a pinprick or a hot or coldstimulus.

Screening Assays

The present invention provides screening methods to identify compoundsthat can inhibit the production or action of BH4. Useful compoundsinclude any agent that can inhibit the biological activity or reduce thecellular level of BH4 or at least one or more than one of any one of theenzymes shown in FIG. 2A. Such enzymes include, for example, GTPcyclohydrolase (GTPCH), Pyruvoyltetrahydropterin (PTPS), SepiapterinReductase (SPR), and Dihydropteridine Reductase (DHPR). As discussedabove, we have shown that DAHP, NAS, and methotrexate are useful totreat, reduce, or prevent pain. Using such agents as lead compounds, forexample, the present screening methods allow the identification ofnovel, specific inhibitors of the BH4 synthetic pathway that function toinduce analgesia. The method of screening may involve high-throughputtechniques.

A number of methods are available for carrying out such screeningassays. According to one approach, candidate compounds are added atvarying concentrations to the culture medium of cells expressing one ormore of the BH4 synthetic enzymes. Gene expression of the BH4 syntheticenzymes is then measured, for example, by standard Northern blotanalysis (Ausubel et al., supra), using any appropriate fragmentprepared from the nucleic acid molecule of the BH4 synthetic enzyme as ahybridization probe or by real time PCR with appropriate primers. Thelevel of gene expression in the presence of the candidate compound iscompared to the level measured in a control culture lacking thecandidate molecule. If desired, the effect of candidate compounds may,in the alternative, be measured at the level of BH4 production using thesame general approach and standard immunological techniques, such asWestern blotting, immunoprecipitation, or immunoassay with an antibodyspecific to the BH4 synthetic enzyme or for BH4 (or its intermediariesor metabolites). For example, immunoassays may be used to detect ormonitor the level of BH4 or GTPCH. Polyclonal or monoclonal antibodieswhich are capable of binding to BH4, BH4 precursors, or BH4 metabolitesmay be used in any standard immunoassay format (e.g., ELISA or RIAassay) to measure the levels of BH4 or its precursors or metabolites.BH4 or its precursors or metabolites can also be measured using massspectroscopy, high performance liquid chromatography, spectrophotometricor fluorometric techniques, or combinations thereof. Total biopterin(BH1, BH2, and BH4) content may further be measured as described furtherbelow.

Alternatively, the screening methods of the invention may be used toidentify candidate compounds that decrease the biological activity ofBH4 by decreasing its binding to BH4-dependent enzymes or BH4-bindingreceptors, or alternatively, that decrease the activity or levels of anyof the BH4 synthetic enzymes. For example, a candidate compound may betested for its ability to decrease GTPCH activity in cells thatnaturally express the enzyme, after transfection with cDNA for theenzyme, or in cell-free solutions containing the enzyme, as describedfurther below. The effect of a candidate compound on the binding oractivation of a BH4-dependent enzyme (such as NOS) or a BH4-bindingreceptor (or analogs) can be tested by radioactive and non-radiaoctivebinding assays, competition assays, enzyme activity assays, receptorsignaling assays.

As a specific example, mammalian cells (e.g., rodent cells) that expressa nucleic acid encoding a BH4 synthetic enzyme are cultured in thepresence of a candidate compound (e.g., a peptide, polypeptide,synthetic organic molecule, naturally occurring organic molecule,nucleic acid molecule, or component thereof). Cells may eitherendogenously express the BH4 synthetic enzyme or may alternatively begenetically engineered by any standard technique known in the art (e.g.,transfection and viral infection) to overexpress the BH4 syntheticenzyme. The expression level of the BH4 synthetic enzyme is measured inthese cells by means of e.g., Western blot analysis and subsequentlycompared to the level of expression of the same protein in control cellsthat have not been contacted by the candidate compound. A compound whichpromotes a decrease in the level of BH4 or intermediary as a result ofreducing its synthesis by reducing the level or activity of one of itssynthetic enzymes is considered useful in the invention. Given itsability to decrease the level or activity of BH4, such a molecule may beused, for example, as an analgesic therapeutic agent to treat, reduce,or prevent pain.

The activity of any of the BH4 synthetic enzymes may be measured by therate at which they consume substrate, e.g., GTP or produce product,e.g., BH4 (see Werner et al. (1996) J Chromatogr B Appl 684:51-58).Radiometric assays are based on the consumption of labeled substrate.For example, GTPCH activity may be assessed by measuring the release oflabeled formic acid originating from a labeled hydrogen atom of GTP andseparation of formic acid from GTP by charcoal (Viveros et al. (1981)Science 213: 349). HPLC-based methods however, are superior to theradioactive method in that HPLC allows determination of the product. Formeasuring GTPCH activity, the tissue or cell homogenate containing GTPCHis incubated with excess GTP (substrate) in the presence of EDTA toensure that the product 7,8 dihydropterin triphosphate is not furthermetabolized by the downstream PTPS which requires Mg²⁺ to operate. Thereaction is stopped by the addition of HCl and iodine. This also resultsin oxidation of the labile 7,8-dihydroneopterin triphosphate to the morestable neopterin triphosphate. Neopterin triphosphate may be analyzeddirectly by ion-pair HPLC and fluorescence detection. Alternatively, themixture is treated with NaOH and alkaline phosphatase to yield neopterinwhich can be analyzed using reversed-phase HPLC with fluorescencedetection, immunoassay or direct fluorescence in case of “pure” samples(such as in vitro kinase assay or CSF).

Sepiapterin reductase activity is commonly assayed using sepiapterin asartificial substrate and measuring levels of total biopterin afteroxidation of BH4 and BH2 to biopterin.

For determination of PTPS activity, the substrate 7,8 dihydroneopterintriphosphate is typically freshly prepared with purified GTPCH. Theincubation mixture also typically contains purified sepiapterinreductase, so that PTPS activity may be evaluated by measuring biopterinlevels after oxidation of BH4 and BH2.

Given its ability to decrease the levels of BH4 or the levels oractivity of a BH4 synthetic enzyme, such a molecule may be used, forexample, as an analgesic therapeutic agent to treat, reduce, or preventpain.

Alternatively, or in addition, candidate compounds may be screened forthose which specifically bind to or inhibit a BH4 synthetic enzyme,BH4-dependent enzyme, or BH4-binding receptor. The efficacy of such acandidate compound is dependent upon its ability to interact with BH4, aBH4 synthetic enzyme or a BH4-binding enzyme or receptor. Such aninteraction can be readily assayed using any number of standard bindingtechniques and functional assays (e.g., those described in Ausubel etal., supra). For example, a candidate compound may be tested in vitrofor interaction and binding with BH4 and its ability to modulate painmay be assayed by any standard assays (e.g., those described herein).

In one particular example, a candidate compound that binds to any of theBH4 synthetic enzymes may be identified using a chromatography-basedtechnique. For example, a recombinant BH4 synthetic enzyme protein maybe purified by standard techniques from cells engineered to express theBH4 synthetic enzymes (e.g., those described above) and may beimmobilized on a column. Alternatively, BH4 may be immobilized on acolumn. A solution of candidate compounds is then passed through thecolumn, and a compound specific for either BH4 or one of the BH4synthetic enzymes is identified on the basis of its ability to bind toBH4 or a BH4 synthetic enzyme and be immobilized on the column. Toisolate the compound, the column is washed to remove non-specificallybound molecules, and the compound of interest is then released from thecolumn and collected. Compounds isolated by this method (or any otherappropriate method) may, if desired, be further purified (e.g., by highperformance liquid chromatography).

Screening for new inhibitors and optimization of lead compounds may beassessed, for example, by assessing GTPCH activity as described above.For screening of multiple substances, a 96 well-based enzyme assay maybe used where purified recombinant GTPCH is incubated together withsubstrate and the potential inhibitor followed by oxidation andmeasurement of neopterin with a fluorescence ELISA reader. Neopterinshows intense fluorescence and can be directly measured.

Assays may also be based on BH4 measurement. BH4 shows no intensefluorescence, because the rings of the molecule are not in the fullyoxidized, aromatic state. To circumvent this, a differential oxidizationmethod in which dihydrobiopterin and BH4 are measured following theiroxidation to biopterin may be used, with a limit of detection of 0.3μmol for biopterin with fluorescence (Fukushima and Nixon, Anal.Biochem. (1980) 102: 176-188). Assays for measuring the activity ofGTPCH or levels of biopterin are described, for example, by Kaneko etal., Brain Res. Brain Res. Protoc. (2001) 8:25-31; Ota et al., J.Neurochem. (1996) 67: 2540-2548; Brautigam et al., Physiol. Chem. (1982)363: 341-343; Curtius et al., Eur. J. Biochem. (1985) 148: 413-419; Steaet al., J. Chromatogr. (1979) 168: 385-393; Werner et al., J.Chromatogr. (1996) 684: 51-58; Werner et al., Methods Enzymol. (1997)281: 53-61; Nagatsu et al., Anal. Biochem. (1981) 110: 182-189; andGeller et al., Biochem Biophys Res Commun (2000) 276: 633-41.

In addition, these candidate compounds may be tested for their abilityto function as analgesic agents (e.g., as described herein). Compoundsisolated by this approach may also be used, for example, as therapeuticsto treat, reduce, or prevent pain. Compounds which are identified asbinding to BH4, any of the BH4 synthetic enzymes, BH4 dependent enzymes,or BH4-binding receptors with an affinity constant less than or equal to10 mM are considered particularly useful in the invention.

Ultimately, the analgesic efficacy of any of the candidate compoundsidentified by the present screening methods may be tested using any ofthe pain models described above.

Potential analgesics include organic molecules, peptides, peptidemimetics, polypeptides, and antibodies that bind to a nucleic acidsequence or polypeptide that encodes any of the BH4 synthetic enzymes orBH4 dependent enzymes or BH4 binding receptors and thereby inhibit orextinguish their activity. Potential analgesics also include smallmolecules that bind to and occupy the binding site of such polypeptidesthereby preventing binding to cellular binding molecules, such thatnormal biological activity is prevented. Other potential analgesicsinclude antisense molecules.

In addition to BH4, any of the BH4 synthetic enzymes may be used uponexpression, as a target for the screening of candidate compounds.Furthermore, each of the compounds provided herein (e.g., DAHP, NAS,methotrexate, BH4, guanine, or any of the compounds found in FIG. 19)may also be used as lead compounds in the discovery and development ofanalgesic compounds.

Test Compounds and Extracts

In general, compounds capable of inducing analgesia are identified fromlarge libraries of both natural products or synthetic (orsemi-synthetic) extracts or chemical libraries according to methodsknown in the art. Those skilled in the field of drug discovery anddevelopment will understand that the precise source of test extracts orcompounds is not critical to the screening procedure(s) of theinvention. Accordingly, virtually any number of chemical extracts orcompounds can be screened using the methods described herein. Examplesof such extracts or compounds include, but are not limited to, plant-,fungal-, prokaryotic- or animal-based extracts, fermentation broths, andsynthetic compounds, as well as modification of existing compounds.Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofchemical compounds, including, but not limited to, saccharide-, lipid-,peptide-, and nucleic acid-based compounds. Synthetic compound librariesare commercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Inaddition, natural and synthetically produced libraries are produced, ifdesired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

In addition, those skilled in the art of drug discovery and developmentreadily understand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their analgesic activity should be employedwhenever possible.

When a crude extract is found to have an analgesic activity, or abinding activity, further fractionation of the positive lead extract isnecessary to isolate chemical constituents responsible for the observedeffect. Thus, the goal of the extraction, fractionation, andpurification process is the careful characterization and identificationof a chemical entity within the crude extract having analgesic activity.Methods of fractionation and purification of such heterogenous extractsare known in the art. If desired, compounds shown to be useful agentsfor the treatment of pain are chemically modified according to methodsknown in the art.

Pharmaceutical Therapeutics

The invention provides a simple means for identifying compounds(including peptides, small molecule inhibitors, and mimetics) capable oftreating, reducing, or preventing pain. Accordingly, a chemical entitydiscovered to have medicinal value using the methods described hereinare useful as either drugs or as information for structural modificationof existing analgesic compounds, e.g., by rational drug design.

For therapeutic uses, the compositions or agents identified using themethods disclosed herein may be administered systemically, for example,formulated in a pharmaceutically-acceptable buffer such as physiologicalsaline. Treatment may be accomplished directly, e.g., by treating theanimal with compounds that treat, reduce, or prevent pain by interferingwith the production of BH4 (by interfering with the biological activityof any of the BH4 synthetic enzymes) or by interfering directly with thebiological activity of BH4 by blocking activation of enzymes that use itas a cofactor or receptors that bind to BH4. Preferable routes ofadministration include, for example, subcutaneous, intravenous,intraperitoneally, intramuscular, or intradermal injections, whichprovide continuous, sustained levels of the drug in the patient.Treatment of human patients or other animals will be carried out using atherapeutically effective amount of an analgesic in aphysiologically-acceptable carrier. Suitable carriers and theirformulation are described, for example, in Remington's PharmaceuticalSciences by E. W. Martin. The amount of the analgesic to be administeredvaries depending upon the manner of administration, the age and bodyweight of the patient, and with the type of disease and extensiveness ofthe disease. Generally, amounts will be in the range of those used forother agents used in the treatment of pain, although in certaininstances lower amounts will be needed because of the increasedspecificity of the compound. A compound is administered at a dosage thatinhibits pain. For example, for systemic administration a compound isadministered typically in the range of 0.1 ng-10 g/kg body weight.

The results of the invention are now described in more detail in thefollowing examples. These examples are provided to illustrate theinvention and should not be construed as limiting.

EXAMPLE 1 Induction of Synthetic Enzymes of the BH4 Pathway byPeripheral Nerve Injury

Transection of the peripheral axons of primary sensory neurons resultsin profound alterations in their metabolism, regenerative capacity,survival, excitability, transmitter function, and sensitivity to diverseextrinsic and intrinsic signals. These changes are mediated bytranscriptional alterations triggered both by the loss of trophicsupport from peripheral target organs and by novel signals generated atthe injury site. These transcriptional changes lead to adaptiveresponses, such as the capacity to survive the injury and re-grow theinjured axon, as well as maladaptive responses that can result in achange in sensation, including the generation of neuropathic pain.

High-density rat oligonucleotide microarrays have been used to detectchanges in gene expression in the dorsal root ganglion (DRG) followingsciatic nerve transection (axotomy). The DRG represents a densecollection of cell bodies of one general class of neurons, the primarysensory neuron. The lesion has a uniform impact on the cells, and theexistence of a large pool of genes with known regulation allows forquality controls for changes identified by the microarrays.

Affymetrix rat U34A oligonucleotide arrays were used to screen forchanges in gene expression in DRG neurons during maturation of the DRGin the embryo and after a sciatic nerve lesion. In the first study wefound that the expression profile of several genes, includingdihydropteridine reductase (DHPR), was characterized by high levels ofexpression early during development, a down-regulation during adulthood,and re-expression following peripheral nerve injury (FIGS. 1A and 1B). Amore detailed study was then performed looking at alterations in geneexpression three days following a peripheral nerve (sciatic) transection(axotomy, Ax) by comparing expression levels with non-injured DRGs(naïve, N) as described previously by Costigan et al., (BMC Neuroscience(2002) 3:16), hereby incorporated by reference. Nine biologicallyindependent array hybridizations were performed (six naïve and threeafter axotomy). DRG tissue (L4 and L5 from the left or ipsilateral sideto the injury) from five male Sprague-Dawley rats was pooled for eachRNA population. Each RNA sample was labeled separately and hybridized toa separate array. Two comparisons were made using two sets of triplicatemicroarrays: naïve versus naïve and naïve versus axotomy. _(G)enes weredefined as detected if they received a present or marginal call in atleast one of the arrays within each comparison. Since each individualsample was pooled from five male Sprague-Dawley animals of a similar ageand from a single supplier (Charles River), biological variation islikely to be minimal.

In addition to DHPR, this analysis further revealed that two othermembers of the tetrahydrobiopeterin synthesis pathway (see FIG. 2A) werealso significantly upregulated by peripheral nerve injury: GTPcyclohydrolase (GTPCH) and sepiapterin reductase (SPR) (See FIG. 3).

EXAMPLE 2 Validation of Microarray Analysis

The induction of BH4 synthetic enzymes by axotomy was next confirmed byvarious methods, such as Northern blot analysis, Northern slot blotanalysis, in situ hybridization, and Western blot analysis. A samplefrom each group was prepared from independent L4 and L5 DRG RNA samplesextracted from different groups of animals than those used for thearrays. FIG. 1B represents a Northern slot blot analysis showing theexpression profile of DHPR during embryonic development and in theadult, before and after axotomy and thereby confirming our microarraydata. Northern Blot analysis to detect GTPCH mRNA levels in naive DRGand 3 days following injury clearly show the marked induction of twotranscripts of 3 kb and 1.2 kb (see FIG. 4A). FIG. 3 summarizes thedegree of the induction of BH4 synthetic enzymes in DRG followingaxotomy. In situ analysis further confirmed the induction of GTPCH mRNAin neurons of the DRG three days post peripheral nerve injury (FIG. 4B).FIGS. 4D-4F represent triplicate Northern blot analysis demonstratingthat the induction of GTPCH, DHPR, and SPR mRNA transcript levels in theDRG following axotomy is sustained for at least 2 weeks. We further showthat the increase in GTPCH mRNA levels following nerve injury isassociated with an increase in protein levels (FIG. 5A) and enzymeactivity (FIG. 5B). FIG. 5A represents a Western Blot analysis of GTPCHprotein levels in naive DRG and 1, 3, 7, and 14 days post-axotomyshowing a marked and sustained increase in GTPCH levels followingaxotomy. GTPCH activity levels in the DRG are markedly higher at sevendays post axotomy relative to control (FIG. 5B). The amount of BH4 inthe DRG is also increased (FIG. 5C) seven days post axotomy relative tocontrol.

EXAMPLE 3 Changes in BH4 Synthetic Enzymes in Neuropathic andInflammatory Pain Models

Triplicate Affymetrix microarrays were used to establish the time courseof changes of expression of the BH4 synthetic pathway members (GTPCH,SPR and DHPR as well as 6-pyruvoyl tetrahydropterin synthase and thefeedback regulatory protein, GTPCH feed-back regulatory protein) in theDRG and in the dorsal horn of the spinal cord in three independentperipheral neuropathic pain models and after peripheral inflammation.GTPCH I, SPR, PTPS and DHPR were all upregulated by a substantial degreeand for prolonged periods in the DRG in all three peripheral neuropathicpain models (FIGS. 6A-6J). The mRNA for all these enzymes was detectablein the dorsal horn (i.e. are constitutively expressed) but showedminimal alterations in expression in the pain models. In both the DRGand dorsal horn, peripheral inflammation also did not produce markedchanges in the constitutive basal level of expression of the BH4synthetic enzymes (FIG. 7A-I).

EXAMPLE 4 Effects of an Inhibitor of GTPCH on Neuropathic Pain

Based on these findings, we hypothesized that the BH4 pathway may have arole in the biological response to peripheral nerve injury includingactivation of cell survival responses, changes in excitability,alterations in transmitter function, and change in growth status. Inparticular, we hypothesized that the pathway may have a role in thegeneration of pain after peripheral nerve injury (peripheral neuropathicpain) for example by increasing NOS activity as a result of the increasein BH4 levels.

To assess whether the BH4 synthetic pathway is involved in neuropathicpain, we examined if DAHP could elicit analgesia in various pain models,such as the spared nerve injury (SNI) model (FIGS. 8A-8H), the chronicconstriction injury (CCI) model (FIGS. 9A and 9B), the formalin assay(FIG. 10), and the CFA model (FIGS. 11A-E).

Using the SNI model, we show that treatment with DAHP (180 mg/kg/dayinjected intraperitoneally) following surgery produced a reduction inmechanical sensitivity (von Frey threshold) and cold pain (coldallodynia by the application of acetone to the paw) relative to ratsinjected with vehicle, whether treatment was initiated at an early timepoint (e.g., three days post-surgery, see FIGS. 8A-8D) or at a latertime point (e.g., seventeen days post-surgery, see FIGS. 8E-8F). Thus,treatment with DAHP could produce analgesia even once neuropathic painwas established. DAHP (6 mg/kg/day i.t.) also reduced mechanical andcold allodynia when administered as a continuous intrathecal infusionthrough a lumbar spinal catheter. The efficacy was comparable withintraperitoneal treatment. The analgesic effects of DAHP were furtherconfirmed in the CCI model (180 mg/kg/d i.p.; FIGS. 9A and 9B). DAHP(single i.p. dose of 180 mg/kg) also reduced the flinching behavior inthe formalin assay (FIG. 10).

The results of FIG. 8 demonstrating the analgesic effects of DAHP in anSNI model were extended through the use of increasing doses of DAHP.FIGS. 22A and 22B demonstrate that a dose-dependent relationship existsbetween the amount of DAHP administered and the nociceptive response tomechanical (von Frey test) or thermal (cold allodynia) stimuli. Thisdose-effect relationship was linear in the dose range tested. Up to thehighest dose of 270 mg/kg/d, no obvious neurological adverse effectswere observed over 14 days of treatment. These results further supportthe pharmacological effect of BH4 pathway inhibitors.

Moreover, as expected, intrathecal administration of BH4 waspro-nociceptive. FIG. 23A demonstrates that intrathecal administrationof BH4 onto the lumbar spinal cord through a chronically implantedcatheter reduces the paw withdrawal latency to a thermal stimulus(Hargreaves model) in naïve rats, indicating an increasedhypersensitivity to heat. Likewise, in rats with pre-existing heathypersensitivity, intrathecal BH4 administration induced heathypersensitivity in the ipsilateral, but not contralateral, paw in aCFA-induced model of paw inflammation.

We next injected complete Freund's adjuvant (CFA) into the right paw ofrats to elicit paw inflammation. We show using the CFA pain model thatDAHP (180 mg/kg i.p.) reduced thermal hyperalgesia, whether treatmentwas initiated 30 min before (FIG. 11A, left side) or 24 h (FIGS. 11A,right side and 11C) after the CFA injection. DAHP had no effect on thepaw withdrawal latency of the non-inflamed contralateral paw (FIG. 11B),indicating that DAHP had no general obvious inhibitory effect on sensoryand motor functions. DAHP (1 mg/kg i.t.) also reduced thermalhyperalgesia when it was delivered to the lumbar spinal cord byintrathecal injection through a lumbar spinal catheter (FIG. 11D).Direct comparison of intraperitoneal and intrathecal DAHP treatmentrevealed that effects are similar with both routes of drugadministration (FIG. 11E) The fact that there was no difference in theanalgesic effect in rats treated with DAHP intrathecally andintraperitoneally infers that DAHP is effective at the level of thespinal cord and DRG.

We next measured the effect of DAHP on inflammatory paw edema. As shownin FIG. 12, measuring the paw weight in the CFA injected paw and thenon-injected control paw showed no difference in the degree of pawinflammation between DAHP treated and control animals. Thus, because theadministration of DAHP has no obvious effect on inflammation (noanti-inflammatory action), our results suggest that DAHP's analgesiceffect is primarily a result of changes in sensory processing in thenervous system

Using the von Frey and the Hargreaves thermal pain test, we further showthat the injection of DAHP in non-injured animals did not result in adifference in motor activity. Based on these results, and the absence ofany detectable changes in locomotion (FIGS. 13A-13B) the possibilitythat DAHP at the doses used has an effect on general sensory and motoractivity seems unlikely. In addition there was not detectable change inthe general level of activity with no obvious signs of sedation.

We next performed pharmacokinetic studies to inspect the levels of DAHPin plasma and CSF (see FIGS. 14A and 14B) and show that the plasmaconcentration rapidly increased after i.p. injection followed by a rapiddistribution in the cerebrospinal fluid. Furthermore, we confirmed thatthe increase in plasma DAHP concentration over time correlated with thebehavioral effect in rats in response to DAHP treatment in the CFA model(FIG. 14C).

EXAMPLE 5 Effects of Other BH4 Inhibitors on Inflammatory or NeuropathicPain

We next evaluated the effect of inhibiting the BH4 synthetic enzymeSepiapterin reductase by administering N-acetyl-serotonin (NAS).Similarly to DAHP, we show that NAS (50 mg/kg i.p.) resulted in areduction of thermal hyperalgesia in the CFA model (see FIG. 15A). NAStreatment also had no effect on the CFA-induced inflammatory paw edema(FIG. 15B).

Similarly, we show that the administration of methotrexate, an inhibitorof DHPR, could result in a reduction in pain in the SNI model inresponse to mechanical and cold allodynia, in the absence of detectableacute toxicity. MTX was administered at low dose systemically (see FIGS.16A and 16B) or by continuous lumbar spinal delivery using an osmoticpump (0.1 mg/kg/d ay)(see FIGS. 16C and 16D). Toxicity was measured asbody weight change over time (see FIGS. 16E and 16F).

Overall, our results demonstrate that the inhibition of BH4 synthesis byadministering DAHP, NAS, or MTX for example, results in analgesia inresponse to thermal, mechanical, and chemical stimuli. Based on theseresults, inhibiting the synthesis of BH4, by reducing the biologicalactivity of BH4 synthetic enzymes for example, induces analgesia, andtherefore, may be used to treat, prevent, or reduce pain in a mammal inneed thereof.

EXAMPLE 6 Molecular and Cellular Effects of DAHP on Neuropathic Pain

Measurement of c-FOS expression in dorsal horn neurons was used as anobjective indication of pain intensity. FIG. 24 demonstrates that c-FOSimmunoreactivity is elevated in ipsilateral dorsal horn neurons ofanimals two hours after receiving formalin injection into the hindpaw. Asignificantly reduced elevation in c-FOS levels were observed in animalsalso administered intraperitoneal DAHP (p<0.05). This indicates thatDAHP acts in the BH4 metabolic pathway upstream of immediate early genec-Fos induction and reduces activation of neurons in the spinal cord.

Apoptosis of dorsal horn neurons contributes to the development ofneuropathic pain following a nerve injury. To further investigate thecellular role of BH4 in neuropathic pain, apoptotic profiles of L4/L5dorsal horn neurons were evaluated using TUNEL staining. FIG. 25demonstrates that intraperitoneal DAHP administration protects dorsalhorn neurons from apoptosis in the SNI model.

As discussed above, BH4 is an essential co-factor for nNOS and iNOSisozymes which have been shown to contribute to pain signaling in thenervous system. We have found that the anti-nociceptive effects of DAHPdo not differ between nNOS knockout mice and wild-type mice (FIG. 26A),demonstrating the nNOS is not essential for the production of theanalgesic effects of DAHP. We have also found that, DAHP induces astronger antinociceptive effect than a high systemic dose of L-NAME, anon-specific NOS inhibitor, and that L-NAME does not further increasethe efficacy of DAHP when injected together (FIG. 26B). Thus, theanti-nociceptive effects of DAHP cannot be attributed merely to aninhibition of NO production and the pro-nociceptive action of BH4 is notmediated solely through a NOS-dependent mechanism. Taken together, thesedata suggest that the pro-nociceptive effects of BH4 are likely mediatedthrough a novel target molecule.

EXAMPLE 7 Localization of GTPCH-I

We have characterized the localization of GTPCH-I. Upregulation of thisenzyme occurs in large and small to medium sized DRG neurons afterperipheral axonal injury (SNI model) compared to unlesioned animals(FIGS. 27A and 27B). Upregulation of GTPCH-I is not, however, observedin the CFA-induced paw inflammation model. Forty to fifty percent ofneurons that upregulate GTPCH-I are also immunoreactive forneurofilament 200 (NF200) which is a marker for neurons havingmyelinated axons (FIG. 29). Thirty to forty percent of GTPCH-I mRNApositive neurons are also immunoreactive for calcitonin gene relatedpeptide (CGRP; FIG. 29). CGRP labels small to medium sized neuropeptidepositive sensory neurons, most of which are nociceptors. GTPCH-Iexpressing neurons do not express nNOS and are not labeled withGriffonia simplicifolia isolectin B4 (IB4; FIG. 29). IB4 labels smallunmyelinated GDNF (glial cell derived neurotrophic factor) responsiveneurons. GTPCH-I-expressing neurons show immunoreactivity for ATF-3which indicates that the upregulation mainly occurs in injured neurons(FIG. 31).

The GTPCH-I transcript is not detectable in the dorsal horn of thespinal cord in either control or SNI-lesioned animals. Isolated injuredmotor neurons show GTPCH-I mRNA when their peripheral axons aretransected by a peripheral nerve injury (FIG. 28A). GTPCH-I feedbackregulatory protein (GFRP) mRNA can be detected in isolated DRG neuronsand its expression does not change after nerve injury (FIG. 28B).

EXAMPLE 8 Upregulation of BH4 Metabolites is Detected in Lesioned DorsalRoot Ganglia

We measured the levels of biopterin and neopterin, stable metabolites ofBH4, in the DRG of animals following SNI lesion. Neopterin is a stablemetabolite of BH4 found following BH4 recycling. The presence ofneopterin may be an index of new BH4 synthesis, whereas biopterin isindicative of BH4 recycling and reuse, but not necessarily newsynthesis. FIG. 30 demonstrates elevated biopterin levels in theipsilateral doral horn (DH), the DRG, and the ScN compared to thecontralateral side. Biopterin increases were reversed in the DH and theScN, but not the DRG, with the administration of DAHP. Neopterin levels,by contrast, were elevated only in the DRG and ScN, but not the DH.These increases were reversed by DAHP administration. Together, thesedata demonstrate the usefulness of measuring stable BH4 metabolites asobjective indicators of pain. Further, these data demonstrate that DAHPinhibits BH4 biosynthesis in vivo and inhibition of the BH4 biosyntheticpathway is a useful mechanism for inducing analgesia.

The above experiments were performed using the following materials andmethods.

Materials and Methods Surgical Procedures

All procedures were performed in accordance with Massachusetts GeneralHospital animal care regulations. Adult male Sprague Dawley rats(200-300 g) were anesthetized with halothane. For the sciatic nervetransection (axotomy), the left sciatic nerve was exposed at the midthigh level, ligated with 3/0 silk, and sectioned distally. The woundwas sutured in two layers, and the animals were allowed to recover. ForSNI the tibial and peroneal branch of the sciatic nerve were ligated andtransected whereas the sural nerve was spared. For CCI, the sciaticnerve was loosely ligated with dexon 4/0 (three ligatures) and for thespinal ligation model the L5 spinal segmental nerve was ligated.

Tissue and RNA Preparation

Animals were terminally anesthetized with CO₂, the L4 and L5 DRGsrapidly removed, and stored at -80° C. Total RNA was extracted fromhomogenized DRG samples using acid phenol extraction (TRIzol reagent,Gibco-BRL). RNA concentration was evaluated by A₂₆₀ measurement andquality assessed by electrophoresis on a 1.5% agarose gel. Each RNAsample used for hybridization of each array was extracted from rat L4and L5 DRGs (10 ganglia pooled from 5 animals, per sample).

Microarray Analysis

Affymetrix rat genome U34A oligonucleotide microarrays, representing8799 known transcripts and expressed sequence tags (ESTs), were usedaccording to the manufacturers instructions (Santa Clara, Calif.http://www.affymetrix.com). Transcript abundance is estimated byanalysis of signal intensity of the probe set for each transcript andcomparison with mismatch controls. The arrays are hybridized withbiotin-labeled cRNA, prepared as per standard Affymetrix protocol.Briefly, total RNA (8 μg) from DRGs was reverse transcribed using anoligo-dT primer coupled to a T7 RNA polymerase binding site.Double-stranded cDNA was made and biotinylated-cRNA synthesized using T7polymerase. The cRNA was hybridized for 16 hours to an array, followedby binding with a streptavidin-conjugated fluorescent marker, and thenincubated with a polyclonal anti-streptavidin antibody coupled tophycoerythrin as an amplification step. Following washing, the chipswere scanned with a Hewlett-Packard GeneArray laser scanner and dataanalyzed using GeneChip software. External standards were included tocontrol for hybridization efficiency and sensitivity.

Hybridization levels for each species of mRNA detected on the arrays areexpressed by intensity (signal) and as present (P), marginal (M) orabsent (A) calls, calculated by Affymetrix software (MAS 5.0, α1=0.04α2=0.06). To normalize the array data standard Affymetrix protocols wereemployed, each array was scaled to a target signal of 2500 across allprobe sets (MAS 5.0).

The arrays were grouped for comparison of a triplicate set of naïve datawith a triplicate post-axotomy set. A probe set was determinedundetected if it received an A call in all of the six arrays involved inthe comparison. Detected were Present or Marginal by MAS5.0 in at leastone array for each analysis. Mean signal and standard deviation werecalculated for each detected probe set. The p-value for rejecting thenull hypothesis that the mean signals were equal between the twotriplicate sets was calculated using an unpaired, two-tailed t-test forindependent samples with unequal variance (Satterthwaite's method).Fold-differences between the mean signals (A and B) in the twotriplicate sets were calculated as max (A, B)/min(A, B) with downregulation relative to naïve expressed as negative.

cDNA Probe production

To generate specific probes for Northern blot hybridization experiments,primers based on the rat accession number provided by Affymetrix weredesigned, primer pairs were chosen using the Primer3 softwarehttp://www-genome.wi.mit.edu/ from the 1000 most 3′ nucleotides withineach accession sequence. PCR was performed on cDNA reverse transcribedfrom total RNA, extracted from lumbar DRGs, using poly-dT as a primer toobtain cDNA fragments (141 to 596 bp). These fragments were subsequentlycloned into the PCRII vector (TA cloning Kit, Invitrogen) and theidentity of each was confirmed by sequencing in both directions. ThesecDNAs were gel-purified and used to produce ³²P-labeled cDNA probes(Prime-It kit, Stratagene).

Northern Blot Analysis

Total RNA was size separated by electrophoresis on a 1.5%agarose/formaldehyde gel (10 μg of total RNA per lane) and transferredto a Hybond N+ nylon membrane. Membranes were hybridized withlabeled-probes (see above) in ExpressHyb (Clontech) overnight at 65° C.,washed, and exposed to X-ray film with an intensifying screen at −80° C.

Slot Blots

Total RNA (1.25 μg) was directly transferred to Hybond N+ nylon membraneunder vacuum using a Hoefer PR648 slot blot apparatus (AmershamPharmacia Biotech). Levels of hybridization were quantified using the24450 phosphorimager system (Molecular Dynamics, Sunnyvale Calif.). Theblots were probed for cyclophilin as a loading control. Loading levelsbetween samples on each blot were normalized using the cyclophilinlevels from the control blot.

Isotopic in Situ Hybridization

DRGs were rapidly removed, embedded in OCT (Tissue Tek), and frozen.Sections were cut serially at 6 μm. Isotopic-in situ hybridization wascarried out using forty-eight base pair oligonucleotide probes, designedto have 50% G-C content and be complementary to the mRNAs whoseaccession numbers were provided by Affymetrix. Probes were 3′-endlabeled with ³⁵S or ³³P-dATP using a terminal transferase reaction andhybridization carried out. Autoradiograms were generated by dippingslides in NTB2 nuclear track emulsion and storing in the dark at 4° C.Sections were exposed for 1-8 weeks (depending on the abundance oftranscript), developed, fixed, and viewed under darkfield using afiber-optic darkfield stage adapter (MVI). Controls to confirmspecificity of oligonucleotide probes included hybridization of sectionswith labeled probe with a 1,000-fold excess of cold probe or labeledprobe with a 1,000-fold excess of another, dissimilar cold probe of thesame length and similar G-C content.

Assays for GTPCH Activity and Biopterin Concentration

Screening for new inhibitors and optimization of lead compounds may beassessed, for example, by assessing GTPCH activity. In this regard, itsinhibition by various chemicals may be determined by incubating theenzyme with GTP and measuring the level of biopterin or neopterinproduction using fluorometric, radiolabel, immunoassay,spectrophotometry, and HPLC techniques.

Using the preferred method, tissue neopterin and biopterin levels weredetermined with a liquid chromatography tandem mass spectrometry method.After acidic pH oxidation with iodine according to Fukushima and Nixon(Methods Enzymol. 66: 429-436, 1980) tissues were extracted by solidphase extraction employing Oasis MCX extraction cartridges andconcentrations of total biopterin, neopterin and the internal standardrhamnopterin were determined by liquid chromatography coupled to tandemmass spectrometry. HPLC analysis was done under gradient conditionsusing a Nucleosil C8 column. MS/MS analyses were performed on an API4000 Q TRAP triple quadrupole mass spectrometer with a Turbo Ion Spraysource. Precursor-to-product ion transitions of m/z 236 192 forbiopterin, m/z 252 192 for neopterin, m/z 265 192 for rhamnopterin wereused for the MRM. Concentrations of the calibration standards, qualitycontrols and samples were evaluated by Analyst software 1.4 (AppliedBiosystems). Linearity of the calibration curve was proven from 0.1-50ng/ml. The coefficient of correlation for all measured sequences was atleast 0.99. The intra-day and inter-day variability was <10%.

Alternative methods to determine BH4 employ radioenzymatic assays thatrequire the production of separate and individual antibodies specificfor each pterin species and/or oxidation state. Separation of pteridinesis accomplished by chromatographic techniques and HPLC. HPLC withfluorescence detection enables rapid and sensitive determination of manybiologically occurring pterins (including biopterin and pterin) in thepicomole range in a single chromatographic run.

Tissue homogenates are centrifuged and the resulting supernatant is usedfor both enzyme and protein assays.

GTP cyclohydrolase I activity is assayed as described by Duch et al.(Mol. Cell Endocrinol. (1986) 47: 209-16) with the followingmodifications. The reaction mixture (500 ml) contained 0.1 M Tris-Cl (pH7.8), 0.3 M KCl, 2.5 mM EDTA, 10% glycerol, 1 mM GTP, and the enzyme.The reaction was carried out at 37° C. for one hour in the dark and wasterminated by the addition of 50 ml of iodine solution (1.0% 12, 2.0% KIin 1.0 N HCl). After keeping the mixture at room temperature for onehour, excess iodine is reduced by addition of 50 ml of 2.0% ascorbate.The mixture was supplemented with 50 ml of 1.0 N NaOH and then incubatedwith 3.0 units (100 ml) of alkaline phosphatase at 37° C. for one hour.The reaction was stopped by the addition of 100 ml of 1.0 N acetic acid.After centrifugation, the supernatant was applied to a Whatman Partisil10 ODS column (4.6×3×250 mm) connected to a Cosmosil 10 C18 column(4.6×3×50 mm). Neopterin was eluted isocratically with a solvent of 50mM sodium acetate buffer (pH 5.0) containing 0.1 mM EDTA and 5% methanolat a flow rate of 0.8 ml/min. The column temperature was maintained at25° C. The eluate was monitored with a fluoromonitor (excitation, 350nm; emission 440 nm). Protein concentration was determined using adye-binding assay kit (Bio-Rad) using immunoglobulin-G as a standard.

Cellular Biopterin Content

Total biopterin (BH1, BH2, and BH4) was measured in tissue lysates afteracidic oxidation of reduced forms of biopterin with iodines. Followingcentrifugation at 12,000 rpm (three times for five minutes each), celllysates are treated with 1% I₂ containing 2% KI in 1N HCl for one hourat 37° C. in the dark. Samples were then centrifuged at 12,000 rpm(three times for five minutes each) and the supernatants treated withascorbate (0.1 M) to remove residual I₂. Extracts were then neutralizedwith 1 N NaOH followed by 200 mM Tris-Cl (pH 7.8). Biopterin wasquantitated by C18 reverse HPLC using an online fluorescence detector.

The determination of cellular biopterin content is described in detailin the following references, Harada et al., Science (1993) 260: 1507-10,Kapatos et al., J. Neurochem. (1999) 72: 669-75, Maita et al., Proc.Natl. Acad. Sci. USA (2002) 99: 1212-7; Moali et al., Chem Res Toxicol(2001)14: 202-10, Rebelo et al., J. Mol. Biol. (2003) 326: 503-16;Renodon-Comiere et al., Biochemistry (1999) 38: 4663-8; Xie et al., J.Biol. Chem. (1998) 273: 21091-8; Yoneyama et al., J. Biol. Chem. (1998)273: 20102-8; Yoneyama et al., Protein Sci. (2001) 10: 871-8; Yoneyamaet al. Arch. Biochem. Biophys. (2001) 388: 67-73, all of which arehereby incorporated by reference.

Dosages

The dosage of individual components or therapeutic combinations of thepresent invention can be readily determined by those skilled in the artof pain management. For example, the dose of an analgesic administeredaccording to the present invention will be the same or less than thatwhich is practiced in the art.

Formulation of Pharmaceutical Compositions

The administration of any compound of this invention may be by anysuitable means that results in a concentration of the compound that iseffective for the treatment of pain. The compound(s) may be contained inany appropriate amount in any suitable carrier substance, and isgenerally present in an amount of 1-95% by weight of the total weight ofthe composition. The composition may be provided in a dosage form thatis suitable for the oral, parenteral (e.g., intravenous, intramuscular,or subcutaneous injection), rectal, or transdermal (topical)administration route. Thus, the composition(s) may be in the form of,e.g., tablets, capsules, pills, powders, granulates, suspensions,emulsions, solutions, gels including hydrogels, pastes, ointments,creams, plasters, drenches, osmotic delivery devices, suppositories,enemas, injectables, or implants. The pharmaceutical compositions may beformulated according to conventional pharmaceutical practice (see, e.g.,Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R.Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulatedto release the active compound (drug) substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain drug action during a predetermined time periodby maintaining a relatively, constant, effective drug level in the bodywith concomitant minimization of undesirable side effects associatedwith fluctuations in the plasma level of the active drug substance(sawtooth kinetic pattern); (iv) formulations that localize drug actionby, e.g., spatial placement of a controlled release composition adjacentto or in the diseased tissue or organ; and (v) formulations that targetdrug action by using carriers or chemical derivatives to deliver thedrug to a particular target cell type.

Administration of compounds in the form of a controlled releaseformulation is especially preferred in cases in which the compound,either alone or in combination, has (i) a narrow therapeutic index(i.e., the difference between the plasma concentration leading toharmful side effects or toxic reactions and the plasma concentrationleading to a therapeutic effect is small; in general, the therapeuticindex, TI, is defined as the ratio of median lethal dose (LD50) tomedian effective dose (ED50)); (ii) a narrow absorption window in thegastro-intestinal tract; or (iii) a very short biological half-life sothat frequent dosing during a day is required in order to sustain theplasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the drug is formulated withappropriate excipients into a pharmaceutical composition that, uponadministration, releases the drug in a controlled manner. Examplesinclude single or multiple unit tablet or capsule compositions, oilsolutions, suspensions, emulsions, microcapsules, microspheres,nanoparticles, patches, and liposomes.

Solid Dosage Forms for Oral Use

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystallinecellulose, starches including potato starch, calcium carbonate, sodiumchloride, lactose, calcium phosphate, calcium sulfate, or sodiumphosphate); granulating and disintegrating agents (e.g., cellulosederivatives including microcrystalline cellulose, starches includingpotato starch, croscarmellose sodium, alginates, or alginic acid);binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drugsubstance in a predetermined pattern (e.g., in order to achieve acontrolled release formulation) or it may be adapted not to release theactive drug substance until after passage of the stomach (entericcoating). The coating may be a sugar coating, a film coating (e.g.,based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or an enteric coating (e.g., based on methacrylic acid copolymer,cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate succinate, polyvinyl acetatephthalate, shellac, and/or ethylcellulose). Furthermore, a time delaymaterial such as, e.g., glyceryl monostearate or glyceryl distearate maybe employed.

The solid tablet compositions may include a coating adapted to protectthe composition from unwanted chemical changes, (e.g., chemicaldegradation prior to the release of the active drug substance). Thecoating may be applied on the solid dosage form in a similar manner asthat described in Encyclopedia of Pharmaceutical Technology, supra.

If more than one drug is administered simultaneously, the drugs may bemixed together in the tablet, or may be partitioned. In one example, afirst drug is contained on the inside of the tablet, and a second drugis on the outside, such that a substantial portion of the second drug isreleased prior to the release of the first drug.

Formulations for oral use may also be presented as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders and granulates may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms

Controlled release compositions for oral use may, e.g., be constructedto release the active drug by controlling the dissolution and/or thediffusion of the active drug substance.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated metylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition containing one or more of the compoundsof the claimed combinations may also be in the form of a buoyant tabletor capsule (i.e., a tablet or capsule that, upon oral administration,floats on top of the gastric content for a certain period of time). Abuoyant tablet formulation of the compound(s) can be prepared bygranulating a mixture of the drug(s) with excipients and 20-75% w/w ofhydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, orhydroxypropylmethylcellulose. The obtained granules can then becompressed into tablets. On contact with the gastric juice, the tabletforms a substantially water-impermeable gel barrier around its surface.This gel barrier takes part in maintaining a density of less than one,thereby allowing the tablet to remain buoyant in the gastric juice.

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of anaqueous suspension by addition of water are convenient dosage forms fororal administration. Formulation as a suspension provides the activeingredient in a mixture with a dispersing or wetting agent, suspendingagent, and one or more preservatives. Suitable dispersing or wettingagents are, for example, naturally-occurring phosphatides (e.g.,lecithin or condensation products of ethylene oxide with a fatty acid, along chain aliphatic alcohol, or a partial ester derived from fattyacids) and a hexitol or a hexitol anhydride (e.g., polyoxyethylenestearate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitanmonooleate, and the like). Suitable suspending agents are, for example,sodium carboxymethylcellulose, methylcellulose, sodium alginate, and thelike.

Parenteral Compositions

The compound(s) may also be administered parenterally by injection,infusion, or implantation (intravenous, intramuscular, subcutaneous, orthe like) in dosage forms, formulations, or via suitable deliverydevices or implants containing conventional, non-toxic pharmaceuticallyacceptable carriers and adjuvants. The formulation and preparation ofsuch compositions are well known to those skilled in the art ofpharmaceutical formulation. Formulations can be found in Remington: TheScience and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in form of a solution, a suspension, an emulsion, aninfusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active drug(s), thecomposition may include suitable parenterally acceptable carriers and/orexcipients. The active drug(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, and/or dispersingagents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable active drug(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, and isotonic sodium chloride solution. The aqueous formulationmay also contain one or more preservatives (e.g., methyl, ethyl orn-propyl p-hydroxybenzoate). In cases where one of the compounds is onlysparingly or slightly soluble in water, a dissolution enhancing orsolubilizing agent can be added, or the solvent may include 10-60% w/wof propylene glycol or the like.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the activedrug(s) may be incorporated in biocompatible carriers, liposomes,nanoparticles, implants, or infusion devices. Materials for use in thepreparation of microspheres and/or microcapsules are, e.g.,biodegradable/bioerodible polymers such as polygalactin, poly-(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamnine) and, poly(lacticacid). Biocompatible carriers that may be used when formulating acontrolled release parenteral formulation are carbohydrates (e.g.,dextrans), proteins (e.g., albumin), lipoproteins, or antibodies.Materials for use in implants can be non-biodegradable (e.g.,polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone),poly(lactic acid), poly(glycolic acid) or poly(ortho esters)).

Rectal Compositions

For rectal application, suitable dosage forms for a composition includesuppositories (emulsion or suspension type), and rectal gelatin capsules(solutions or suspensions). In a typical suppository formulation, theactive drug(s) are combined with an appropriate pharmaceuticallyacceptable suppository base such as cocoa butter, esterified fattyacids, glycerinated gelatin, and various water-soluble or dispersiblebases like polyethylene glycols and polvoxyethylene sorbitan fatty acidesters. Various additives, enhancers, or surfactants may beincorporated.

Percutaneous and Topical Compositions

The pharmaceutical compositions may also be administered topically onthe skin for percutaneous (transdermal) absorption in dosage forms orformulations containing conventionally non-toxic pharmaceuticalacceptable carriers and excipients including microspheres and liposomes.The formulations include creams, ointments, lotions, liniments, gels,hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters, andother kinds of transdermal drug delivery systems. The pharmaceuticallyacceptable carriers or excipients may include emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

Examples of emulsifying agents are naturally occurring gums (e.g., gumacacia or gum tragacanth) and naturally occurring phosphatides (e.g.,soybean lecithin and sorbitan monooleate derivatives). Examples ofantioxidants are butylated hydroxy anisole (BHA), ascorbic acid andderivatives thereof, tocopherol and derivatives thereof, butylatedhydroxy anisole, and cysteine. Examples of preservatives are parabens,such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride.Examples of humectants are glycerin, propylene glycol, sorbitol, andurea. Examples of penetration enhancers are propylene glycol, DMSO,triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide,2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol, andAZONE™. Examples of chelating agents are sodium EDTA, citric acid, andphosphoric acid. Examples of gel forming agents are CARBOPOL™, cellulosederivatives, bentonite, alginates, gelatin and polyvinylpyrrolidone.Examples of ointment bases are beeswax, paraffin, cetyl palmitate,vegetable oils, sorbitan esters of fatty acids (Span), polyethyleneglycols, and condensation products between sorbitan esters of fattyacids and ethylene oxide (e.g., polyoxyethylene sorbitan monooleate(TWEEN™)).

The pharmaceutical compositions described above may be applied by meansof special drug delivery devices such as dressings or alternativelyplasters, pads, sponges, strips, or other forms of suitable flexiblematerial.

Controlled Release Percutaneous and Topical Compositions

There are several approaches for providing rate control over the releaseand transdermal permeation of a drug, including: membrane-moderatedsystems, adhesive diffusion-controlled systems, matrix dispersion-typesystems, and microreservoir systems. A controlled release percutaneousand/or topical composition may be obtained by using a suitable mixtureof the above-mentioned approaches.

In a membrane-moderated system, the active drug is present in areservoir which is totally encapsulated in a shallow compartment moldedfrom a drug-impermeable laminate, such as a metallic plastic laminate,and a rate-controlling polymeric membrane such as a microporous or anon-porous polymeric membrane (e.g., ethylene-vinyl acetate copolymer).The active compound is only released through the rate-controllingpolymeric membrane. In the drug reservoir, the active drug substance mayeither be dispersed in a solid polymer matrix or suspended in a viscousliquid medium such as silicone fluid. On the external surface of thepolymeric membrane, a thin layer of an adhesive polymer is applied toachieve an intimate contact of the transdermal system with the skinsurface. The adhesive polymer is preferably a hypoallergenic polymerthat is compatible with the active drug.

In an adhesive diffusion-controlled system, a reservoir of the activedrug is formed by directly dispersing the active drug in an adhesivepolymer and then spreading the adhesive containing the active drug ontoa flat sheet of substantially drug-impermeable metallic plastic backingto form a thin drug reservoir layer.

A matrix dispersion-type system is characterized in that a reservoir ofthe active drug substance is formed by substantially homogeneouslydispersing the active drug substance in a hydrophilic or lipophilicpolymer matrix and then molding the drug-containing polymer into a discwith a substantially well-defined surface area and thickness. Theadhesive polymer is spread along the circumference to form a strip ofadhesive around the disc.

In a microreservoir system, the reservoir of the active substance isformed by first suspending the drug solids in an aqueous solution ofwater-soluble polymer, and then dispersing the drug suspension in alipophilic polymer to form a plurality of microscopic spheres of drugreservoirs.

Other Embodiments

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

Other embodiments are within the claims.

1. A method of treating, reducing, or preventing pain or theconsequences or development of a peripheral nerve lesion in a mammal,said method comprising administering to said mammal a composition thatreduces the tetrahydrobiopterin (BH4) biological activity in an amountsufficient to treat, reduce, or prevent pain or the exacerbation of aperipheral nerve lesion due to overproduction of BH4.
 2. The method ofclaim 1, wherein said mammal is a human.
 3. The method of claim 1,wherein the pain is reduced by reducing the BH4 levels in primarysensory neurons or dorsal horn neurons.
 4. The method of claim 3,wherein said primary sensory neurons are in a dorsal root ganglion or atrigeminal ganglion.
 5. The method of claim 4, wherein said dorsal hornneurons are in the spinal cord or spinal nucleus of the trigeminal inthe brainstem.
 6. The method of claim 1, wherein the reduction in BH4biological activity is the result of a reduction in BH4 synthesis orrecycling.
 7. The method of claim 1, wherein said BH4 biologicalactivity is reduced by increasing the expression, GTPCH-binding, oractivity of GTP cyclohydrolase feedback regulatory protein (GFRP). 8.The method of claim 6, wherein said reduction in BH4 synthesis is theresult of a reduction in the level or biological activity of at leastone enzyme selected from the group consisting of sepiapterin reductase(SPR), Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase(GTPCH), Pterin-4α-carbinolamine dehydratase, and dihydropteridinereductase (DHPR).
 9. The method of claim 8, wherein said reduction inBH4 synthesis is the result of a reduction in the biological activity ofat least one enzyme selected from the group consisting of sepiapterinreductase (SPR), GTP cyclohydrolase (GTPCH), and dihydropteridinereductase (DHPR).
 10. The method of claim 8, wherein the biologicalactivity of at least two of said enzymes is reduced.
 11. The method ofclaim 10, wherein the biological activity of at least three of saidenzymes is reduced.
 12. The method of claim 8, wherein said biologicalactivity is reduced by at least 10%.
 13. The method of claim 12, whereinsaid biological activity is reduced by at least 40%.
 14. The method ofclaim 1, wherein said composition comprises methotrexate.
 15. The methodof claim 1, wherein said composition comprises at least one compoundselected from the group consisting of 2,4 diamino 6-hydroxypyrimidine(DAHP), Tetrahydro-L-biopterin, L-Sepiapterin, 7,8-dihydro-L-Biopterin,6,7-dimethyltetrahydropterin hydrochloride, and 8-bromo-cGMP.
 16. Themethod of claim 1, wherein said composition comprises at least onecompound selected from the group consisting of N-acetyl-serotonin (NAS),N-Chloroacetylserotonin, N-Methoxyacetylserotonin, andN-Chloroacetyldopamine.
 17. The method of claim 1, wherein saidcomposition comprises a compound having the formula:

wherein R¹ is H, C₁₋₆ alkyl, halo, NO₂, CN, CO₂R⁴, CONR⁴R⁵, SO₂R⁴,SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵, wherein each of R⁴ and R⁵ is, independently, H,C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl,R² is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl, and R³ is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶, CONR⁷R⁸, SO₂R⁶, or SO₂NR⁷R⁸, whereinR⁶ is C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄alkheteroaryl and each of R⁷ and R⁸ is, independently, H, C₁₋₆ alkyl,C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl; R³ is asabove and R¹ and R² together are represented by

wherein the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl; R³ is as above and R¹ and R² together arerepresented by

wherein the N of the R¹/R² linkage forms a bond to the pyrimidinonering, each of R¹² and R¹³ is as above, and R¹⁴ is OR⁴, halo, NO₂, CN,CO₂R⁷, CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, wherein each of R⁴, R⁷ and R⁸ is,independently, H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl; or R¹ and R² together are represented by

wherein the N of the R¹/R² linkage forms a bond to the pyrimidinonering, each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, anda double bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².
 18. The method of claim 17, wherein said compositioncomprises a compound of formula (I), wherein R¹ is H, C₁₋₆ alkyl, halo,NO₂, CN, CO₂R⁴, CONR⁴R⁵, SO₂R⁴, SO₂NR⁴R⁵, OR⁴, or NR⁴R⁵, wherein each ofR⁴ and R⁵ is, independently, H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl, R² is H, C₁₋₆ alkyl, C₆₋₁₂ aryl,heteroaryl, C₁₋₄ alkaryl, or C_(i-4) alkheteroaryl, and R³ is H, C₁₋₆alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R⁶,CONR⁷R⁸, SO₂R⁶, or SO₂NR⁷R⁸, wherein R⁶ is C₁₋₆ alkyl, C₆₋₁₂ aryl,heteroaryl, C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl and each of R⁷ and R⁸is, independently, H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl,or C₁₋₄ alkheteroaryl.
 19. The method of claim 1, wherein saidcomposition comprises a compound of formula:

wherein R¹ and R² together are represented by

wherein the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, alkaryl, or C₁₋₄alkheteroaryl.
 20. The method of claim 1, wherein said compositioncomprises a compound of formula:

wherein R³ is as above and R¹ and R² together are represented by

wherein the N of the R¹/R² linkage forms a bond to the pyrimidinonering, each of R¹² and R¹³ is as above, and R¹⁴ is OR⁴, halo, NO₂, CN,CO₂R⁷, CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, wherein each of R⁴, R⁷ and R⁸ is,independently, H, C₁-C₆ alkyl, C₆₋₁₂ C₁₂ aryl, C₁-C₄ alkaryl,heteroaryl, or C₁-C₁ alkheteroaryl.
 21. The method of claim 1, whereinsaid composition comprises a compound of formula:

wherein R¹ and R² together are represented by

wherein the N of the R¹/R² linkage forms a bond to the pyrimidinonering, each of R⁹,R¹⁰, R¹¹, R¹², and R¹⁴ are as above, R³ does not exist,and a double bond is formed between the carbon bearing R¹⁴ and thenitrogen bearing R².
 22. The method of claim 1, wherein said compositioncomprises a compound of formula:

wherein R¹ and R² together are represented by

wherein the N, O, or S of the R¹/R² linkage forms a bond to thepyrimidinone ring and each of R⁹, R¹⁰, R¹¹, R¹², and R¹³ is,independently H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.
 23. The method of claim 1, wherein said compositioncomprises a compound of formula:

wherein R³ is as above and R¹ and R² together are represented by

wherein the N of the R¹/R² linkage forms a bond to the pyrimidinonering, each of R¹² and R¹³ is as above, and R¹⁴ is OR⁴, halo, NO₂, CN,CO₂R⁷, CONR⁷R⁸, SO₂R⁷, SO₂NR⁷R⁸, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, or C₁₋₄ alkheteroaryl, wherein each of R⁴, R⁷ and R⁸ is,independently, H, C₁-C₆ alkyl, C₆-C₁₂ aryl, C₁-C₄ alkaryl, heteroaryl,or C₁-C₄ alkheteroaryl.
 24. The method of claim 1, wherein saidcomposition comprises a compound of formula:

wherein R¹ and R² together are represented by

wherein the N of the R¹/R² linkage forms a bond to the pyrimidinonering, each of R⁹, R¹⁰, R¹¹, and R¹⁴ are as above, R³ does not exist, anda double bond is formed between the carbon bearing R¹⁴ and the nitrogenbearing R².
 25. The method of claim 18, wherein said compositioncomprises a compound selected from the group consisting of


26. The method of claim 1, wherein said composition comprises a compoundhaving the formula:

wherein R¹⁵ is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl; and R¹⁶ is H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl,C₁₋₄ alkaryl, C₁₋₄ alkheteroaryl, CO₂R¹⁷, CONR¹⁸R¹⁹, SO₂R¹⁷ orSO₂NR¹⁸R¹⁹, wherein R¹⁷ is C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄alkaryl, or C₁₋₄ alkheteroaryl and each of R¹⁸ and R¹⁹ is,independently, H, C₁₋₆ alkyl, C₆₋₁₂ aryl, heteroaryl, C₁₋₄ alkaryl, orC₁₋₄ alkheteroaryl.
 27. The method of claim 1, wherein said pain isacute pain.
 28. The method of claim 1, wherein said pain is chronicpain.
 29. The method of claim 1, wherein said pain is selected from thegroup consisting of peripheral and central neuropathic pain,inflammatory pain, functional pain nociceptive pain, and headache. 30.The method of claim 1, further comprising a second therapeutic agent.31. The method of claim 30, wherein said second therapeutic agent is ananalgesic agent.
 32. The method of claim 30, wherein said analgesicagent is a non-steroidal anti-inflammatory agent (NSAIDs), opioidreceptor agonist, tricyclic antidepressant, SSRI, anticonvulsant,clonidine, sodium or calcium channel blocker, potassium channel opener,5-HT1D receptor agonist.
 33. The method of claim 32, wherein said secondtherapeutic agent is an inhibitor of an enzyme selected from the groupconsisting of nitric oxide synthase (NOS), tyrosine hydroxylase,tryptophan hydroxylase I (non-neuronal TphI), tryptophan hydroxylase II(neuronal Tph II), phenylalanine hydroxylase, dopamine-β-hydroxylase,N-methyltransferase, and ether lipid oxidase.
 34. The method of claim30, wherein said therapeutic agent that reduces the levels oftetrahydrobiopterin (BH4) and said second therapeutic agent areadministered within one hour of each other.
 35. The method of claim 30,wherein said therapeutic agent that reduces the levels oftetrahydrobiopterin (BH4) and said second therapeutic agent areadministered simultaneously.
 36. The method of claim 30, wherein saidtherapeutic agent that reduces the levels of tetrahydrobiopterin (BH4)and said second analgesia-inducing compound are administered in the samepharmaceutical formulation.
 37. A method of diagnosing pain or aperipheral nerve lesion in a mammal, said method comprising detecting anincrease in BH4, BH4 metabolite, BH4 precursor, or BH4 intermediate in abiological sample from said mammal.
 38. The method of claim 37, whereinsaid BH4 metabolite is pterin, biopterin, 7,8 dihydropterin,7,8-dihydroxanthopterin, xanthopterin, isoxanthopterin, leucopterin,7,8-dihydroneopterin, or neopterin.
 39. The method of claim 37, whereinsaid BH4 intermediate is 7,8-dihydroneopterin triphosphate, neopterin,or 6-pyruvoyl tetrahydropterin.
 40. The method of claim 37, wherein saidbiological sample is selected from the group consisting of blood, serum,plasma, tissue sample, urine, cerebrospinal fluid, synovial fluid,tissue exudate, or tissue sample.
 41. The method of claim 37, whereinsaid increase is at least a 20% increase relative to control conditions.42. A method of diagnosing pain or a peripheral nerve lesion in amammal, said method comprising detecting an increase in the activity orlevel of a BH4 synthetic enzyme in primary sensory neurons or dorsalhorn neurons of said mammal.
 43. The method of claim 42, wherein saidBH4 synthetic enzyme is selected from the group consisting ofsepiapterin reductase (SPR), Pyruvoyltetrahydropterin Synthase (PTPS),GTP cyclohydrolase (GTPCH), Pterin-4α-carbinolamine dehydratase, anddihydropteridine reductase (DHPR).
 44. The method of claim 42, whereinsaid detecting is performed by imaging techniques.
 45. The method ofclaim 44, wherein said imaging is positron emission tomography (PET).46. The method of claim 42, wherein said increase is at least a 20%increase relative to control conditions.